The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gurney, K. B.
Right arrow Articles by Uittenbogaart, C. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gurney, K. B.
Right arrow Articles by Uittenbogaart, C. H.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 2002, 169: 5338-5346.
Copyright © 2002 by The American Association of Immunologists

TCR{gamma}{delta}+ and CD161+ Thymocytes Express HIV-1 in the SCID-hu Mouse, Potentially Contributing to Immune Dysfunction in HIV Infection1

Kevin B. Gurney*, Otto O. Yang, S. Brian Wilson|| and Christel H. Uittenbogaart2,*,{dagger},{ddagger},§

Departments of * Microbiology, Immunology, and Molecular Genetics and {dagger} Pediatrics, {ddagger} Jonnson Comprehensive Cancer Center, § University of California, Los Angeles AIDS Institute, and Division of Infectious Diseases, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095; and || Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The vast diversity of the T cell repertoire renders the adaptive immune response capable of recognizing a broad spectrum of potential antigenic peptides. However, certain T cell rearrangements are conserved for recognition of specific pathogens, as is the case for TCR{gamma}{delta} cells. In addition, an immunoregulatory class of T cells expressing the NK receptor protein 1A (CD161) responds to nonpeptide Ags presented on the MHC-like CD1d molecule. The effect of HIV-1 infection on these specialized T cells in the thymus was studied using the SCID-hu mouse model. We were able to identify CD161-expressing CD3+ cells but not the CD1d-restricted invariant V{alpha}24/V{beta}11/CD161+ NK T cells in the thymus. A subset of TCR{gamma}{delta} cells and CD161-expressing thymocytes express CD4, CXCR4, and CCR5 during development in the thymus and are susceptible to HIV-1 infection. TCR{gamma}{delta} thymocytes were productively infectable by both X4 and R5 virus, and thymic HIV-1 infection induced depletion of CD4+ TCR{gamma}{delta} cells. Similarly, CD4+CD161+ thymocytes were depleted by thymic HIV-1 infection, leading to enrichment of CD4-CD161+ thymocytes. Furthermore, compared with the general CD4-negative thymocyte population, CD4-CD161+ NK T thymocytes exhibited as much as a 27-fold lower frequency of virus-expressing cells. We conclude that HIV-1 infection and/or disruption of cells important in both innate and acquired immunity may contribute to the overall immune dysfunction seen in HIV-1 disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
As the primary site of T cell development, the thymus plays a critical role in producing functionally responsive, self-tolerant CD4 Th cells and CD8 T cytotoxic cells. The thymus is readily infected by HIV-1 both in vivo and in vitro (1, 2, 3, 4, 5, 6). In vivo, HIV-1 likely gains access to the thymus soon after infection, as inferred from simian immunodeficiency virus infection in macaques (7). Studying thymic infection by HIV-1 in vitro, we and others have documented the greater capacity for X4 virus infection and replication in the thymus over R5 viruses due to greater percentage of CXCR4 coreceptor expression than CCR5 coreceptor expression (8, 9, 10). Furthermore, X4 viruses replicate efficiently in the CXCR4+/high immature thymocytes in the cortex, whereas R5 viruses replicate much slower and primarily in the more mature thymocytes (9, 11).

Thymic stroma is uniquely suited by expression of both MHC class I and class II genes to direct the development of MHC-restricted T cells. However, certain T cell gene rearrangements impart T cell recognition to other nonclassical MHC-like molecules. The CD1 family of molecules is nonpolymorphic and present nonpeptide Ags and self-Ags to specialized T cells. The V{delta}1 subset of TCR{gamma}{delta} cells recognize nonpeptide Ags in the context of CD1c molecules and the MHC-related MIC proteins (12, 13), and certain T cells that typically express the NK receptor protein 1 (NKRP13; CD161) interact with glycolipid Ags on the CD1d molecule (14). These cell types possess specialized functions that contribute to both innate and acquired immunity.

TCR{gamma}{delta} cells are particularly abundant in the intestinal mucosa and are found in other epithelial tissues and peripheral blood (15). These cells exhibit a more restricted repertoire compared with TCR{alpha}{beta} cells and serve as a link between innate and adaptive immunity by interaction with specific pathogens such as mycobacteria (16, 17). The thymus, cord blood, and epithelial tissues contain greater levels of TCR{gamma}{delta} cells expressing V{delta}1 than V{delta}2 (15, 18, 19). In the adult peripheral blood, V{delta}2 cells represent the dominant population (70%) (18), perhaps due to peripheral activation and expansion of V{delta}2 cells after exposure to Ag (30–60%) (15, 18, 19).

However, in HIV-1-infected individuals there is a shift in the proportions of peripheral blood V{delta} subsets, with a drop in the proportion of the dominant V{delta}2+ cells and a rise in V{delta}1+ cells (20, 21). Since only 1–5% of peripheral TCR{gamma}{delta} cells express CD4, direct HIV infection and disruption of the peripheral TCR{gamma}{delta} pool is unlikely to be the explanation (22). However, up to 70% of TCR{gamma}{delta} cells in the thymus express CD4 (23), raising the possibility that HIV-1 infection of the thymus may explain the changes in the peripheral TCR{gamma}{delta} populations. Since TCR{gamma}{delta} cells are exported to the periphery, understanding the effects of HIV-1 in the thymus may help in understanding the phenotype of TCR{gamma}{delta} cells in the periphery.

Other specialized T cells are T cells expressing NK cell-associated markers, namely, NKRP1A (CD161). A subset of these cells express the invariant V{alpha}24/J{alpha}Q TCR that recognizes glycolipid Ag presented on the MHC-related CD1d molecule (14). The basis for their immunoregulatory role is believed to be the abundant secretion of both Th1 and Th2 cytokines, IFN-{gamma} and IL-4, respectively, upon TCR ligation (24, 25). In addition, activation-induced expression of macrophage-inflammatory protein 1{alpha} and macrophage-inflammatory protein 1{beta} signals the recruitment of and interaction with CD1d-expressing myeloid dendritic cells (26). They also exhibit an activated phenotype based on CD69 expression even at birth (27, 28) and possess cytolytic activity (24, 25). These specialized effector functions have been described as playing a role in antitumor responses, autoimmune responses, and antimicrobial responses (for review, see Ref. 29). Recently, van der Vliet et al. (30) reported that these invariant NK T cells are reduced in HIV-1-infected individuals. The decreased cell number was attributed to apoptosis mediated by abundant Fas expression on these cells (30).

CD1d restriction is not limited to the invariant NK T cell population, because CD1d restriction has been found on T cells expressing the NKRP1 (CD161) with diverse repertoire (31). Nor is CD161 expression exclusive for NK T cells, for as much as 25% of the peripheral blood T cells express CD161 (32) where invariant NK T cells make up <0.1% (33). Furthermore, CD161 and other NK markers are expressed on activated CD8+ CTL in both humans and mice (for review, see Ref. 34). This C-type lectin expressed on NK T cells has costimulatory activity (35), which may also be the same for activated CTL. In HIV-1 infection, the number of CD28-CD8+ peripheral T cells expressing NK markers such as CD161 is increased (36), likely due to the chronic activation state of the immune system as observed in other viral diseases, autoimmunity, cancers, and in the elderly (37).

The present study was undertaken to investigate whether TCR{gamma}{delta} thymocytes and CD161+ thymocytes can be productively infected in the SCID-hu mouse model. Due to our inability to identify the canonical NK T cell phenotype (V{alpha}24/J{alpha}Q/CD161) in the human thymus, we studied the total CD161+/CD3+ thymocyte population. HIV-1 thymic infection could potentially disrupt the function of these cells and contribute to the observed changes of these cells in the periphery, leading to immunodeficiency.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and mAbs

Monoclonal Abs to CD3, CD4, CD8, TCR{gamma}{delta}, CD161, and isotype control Abs mouse IgG1 and mouse IgG2 conjugated with fluorescein (FITC), PE, and/or allophycocyanin were obtained from BD Immunocytometry Systems (San Jose, CA). The mAbs KC57-FITC and KC57-PE (which identify intracellular HIV p17 gag and precursor gag Ag expression) and TCRV{alpha}24 and TCRV{beta}11 were obtained from Beckman Coulter (Miami, FL). Monoclonal Abs to CD3, CD4, and CD8 conjugated with Tricolor (Cy5-PE-tandem, referred as TC) and the mouse IgG2a control Ab conjugated to TC were obtained from Caltag Laboratories (Burlingame, CA). Monoclonal Abs to CCR5 and CXCR4 conjugated to FITC, PE, or allophycocyanin were obtained from BD PharMingen (La Jolla, CA). mAb, clone 6B11-FITC, raised to the CDR3 region of V{alpha}24+ NK T cells was a gift from Dr. S. B. Wilson (Dana-Farber Cancer Institute, Boston, MA). CD1d tetramers both loaded and unloaded with the {alpha}-galactosylceramide ligand were gifts from Dr. M. Kronenberg (La Jolla Institute for Allergy and Immunology, La Jolla, CA). 7-Amino-actinomycin D and Tween 20 were obtained from Sigma-Aldrich (St. Louis, MO). Actinomycin D was obtained from Boehringer Mannheim (Indianapolis, IN). Paraformaldehyde was obtained from Polysciences (Warrington, PA). Recombinant human IL-2 (1.5 x 106 U/ml) and IL-4 (0.7 mg/ml) was provided by Amgen (Thousand Oaks, CA). Recombinant human IL-15 (1.9 mg/ml) was obtained from Amgen (Thousand Oaks, CA).

Thymocyte and PBMC preparation

Normal human postnatal thymus specimens were obtained from infants and children undergoing corrective cardiac surgery. The tissue was cut into small pieces and RBC were removed by NH4Cl-Tris lysis and passed over a cell strainer to generate a single-cell suspension of thymocytes as previously described (8). The cells were washed in PBS and serum-free medium (AT-IMDM) consisting of IMDM (Omega Scientific, Tarzana, CA) supplemented with delipidated BSA (Sigma-Aldrich) at 1100 µg/ml, transferrin (Sigma-Aldrich) at 85 µg/ml, 2 mM glutamine, and penicillin/streptomycin at 25 U/25 µg/ml (8, 38). The thymocytes were resuspended at 2 x 105 cells/ml in AT-IMDM for HIV infection (see below).

PBMC were obtained from the Virology Core Facility at the University of California, Los Angeles. Briefly, blood was drawn from normal donors and spun over Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ) gradient. Cells in the buffy coat were collected and counted before flow cytometric staining.

TCR{gamma}{delta} thymocyte purification

TCR{gamma}{delta} thymocytes from the postnatal thymus specimens were purified using the TCR{gamma}{delta} microbead kit by MACS with positive selection columns on a Variomax magnet according to the manufacturer’s guidelines (Miltenyi Biotec, Auburn CA) to >75% purity determined by flow cytometric staining.

HIV-1 infection of SCID-hu mice and postnatal thymocytes

The syncytium-inducing, CXCR4 tropic hybrid molecular clone HIV-1NL4-3 (NL4-3) was used for part of these studies (39). Virus stocks were prepared from 24-h harvests of supernatants from CEM cells (CCRF-CEM) infected with virus derived from COS cells electroporated with plasmid pNL4-3 (39). The nonsyncytium-inducing CCR5-tropic molecular clone HIV-1JR-CSF (JR-CSF) stocks were prepared from 24-h harvests of supernatants from stimulated PBMC infected with the supernatant of COS cells electroporated with plasmid pYKJR-CSF (40) and expanded in stimulated PBMC or in a cryopreserved pool of purified activated allogenic CD4+ cells prepared as described in the literature (41). Briefly, allogenic CD4+ cells from three normal donors were individually purified by capture in CD4 mAb-coated tissue culture flasks (Applied Immunosciences, Santa Clara, CA) and activated by stimulation with Ab to CD3 (OKT3; Ortho Biotech, Raritan, NJ) at 200 ng/ml and with rIL-2 (5000 U/ml) for 5 days. Cells from three donors were combined, cryopreserved in liquid nitrogen, and then thawed and cultured in medium with IL-2 for 2–3 days before infection. Virus stocks were stored at -70°C and treated with 2 µg/ml DNase (Worthington Biochemical, Lakewood, NJ) for 30 min at room temperature in the presence of 0.01 M MgCl2 before infections. All infections were standardized by determining infectious units (i.u.) in limiting dilution studies using PHA-stimulated PBMC (42, 43).

C.B.17 SCID mice were bred at the University of California, Los Angeles and implanted with human fetal thymus and liver graft (thy/liv) under the murine kidney capsule as previously described (44, 45, 46, 47). Four to 6 mo postimplantation, the thy/liv grafts were infected by direct injection of HIV into the graft. Ten nanograms of p24 from R5 HIV-1 JR-CSF and 2 ng of p24 X4 HIV-1 NL4-3 were injected in the implants in a 50-µl volume, equivalent to 10,000 i.u. per implant and 100 i.u. per implant, respectively. Mock-infected implants, used as controls in all experiments, were injected with an equal volume of the appropriate control supernatant from uninfected cells used to grow the virus. At 4, 5, 7, and 8 wk postinfection, mice were sacrificed and the thy/liv implant was processed as described above for the postnatal thymus specimens.

TCR{gamma}{delta} thymocytes, TCR{gamma}{delta}-depleted thymocytes, and total thymocytes were infected in vitro and cultured as previously described for total thymocytes (8, 38). Briefly, 2 x 105 freshly isolated, nonstimulated thymocytes were incubated with 7 ng of viral p24 in the presence of 10 µg/ml polybrene (Sigma-Aldrich) for 1 h at 37°C. Control thymocytes were mock infected in the presence of polybrene with supernatants from the same uninfected cells used to prepare the virus stocks. After infection, the cells were washed and resuspended in serum-free medium in the presence of the cytokine IL-4 (20 ng/ml). Viral replication was assessed by measuring viral p24 Ag in the supernatant by ELISA (Beckman Coulter)

Immunofluorescent intracellular staining and flow cytometry

Surface and intracellular immunophenotyping of purified human thymocytes and PBMC with directly conjugated Abs was performed as previously described (48, 49). Briefly, for intracellular staining, cells were surface immunophenotyped, fixed in 1% paraformaldehyde, and subsequently permeabilized in 0.2% Tween 20 for 15 min at 37°C. The cells were washed with PBS containing 2% newborn calf serum and 0.1% sodium azide (FACS buffer), blocked with human AB serum, and stained with 2.5 µl of KC57 fluorescent Ab or IgG control. Finally, cells were washed with 0.2% Tween 20 and resuspended in FACS buffer before acquisition on a dual-laser FACSCalibur flow cytometer (BD Immunocytometry Systems). From 10,000 to 600,000 events were acquired on each sample. Multiparameter data acquisition and analysis was performed with CellQuest software (BD Immunocytometry Systems).

Statistics

The unpaired two-tailed Student’s t test with unequal variance was used to compare differences in CD4 expression between fetal and postnatal CD161+CD3+ thymocytes and p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TCR {gamma}{delta}+ cells in the thymus express CD4 and coreceptors for HIV-1

To determine whether TCR{gamma}{delta} cells in the thymus are targets for HIV-1 infection with X4 and R5 virus, we investigated the expression levels of the appropriate receptors for entry on TCR{gamma}{delta} cells from postnatal and fetal thymus specimens. We found that in contrast to peripheral TCR{gamma}{delta} cells that lack CD4, 30–85% (mean, 45 ± 14%) of the TCR{gamma}{delta} cells in the thymus expressed CD4 in 25 different fetal and postnatal thymus specimens. A representative example is shown in Fig. 1Go. CXCR4 expression was found on 70–100% (mean 85 ± 12%) of TCR{gamma}{delta} cells from 10 different fetal and postnatal thymus specimens (Fig. 1Go). Thus, as with the total thymocyte population, CXCR4 is expressed on almost all cells, yet there are relatively fewer X4 HIV-1 target cells due to the lower percentage of CD4-expressing cells in the TCR{gamma}{delta} population than in the total thymocyte population. In contrast, there are more R5 HIV target cells in the TCR{gamma}{delta} population than the total population due to greater CCR5 expression (1–7% of TCR{gamma}{delta} thymocytes; Fig. 1Go) vs <1% in the total population as previously reported (8). These data indicate that fetal as well as postnatal TCR{gamma}{delta}+ cells are potential targets for HIV-1 infection. Therefore, experiments were designed to answer the question whether TCR{gamma}{delta}+ cells are infected by HIV-1 in vivo, in the SCID-hu mouse, and in vitro by X4 and R5 viruses.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 1. CD4, CCR5, and CXCR4 expression on TCR{gamma}{delta}+ cells. Four-color flow cytometric analysis of fetal thymocytes (A–C) and PBMC gated on the lymphocyte gate (D–F). Cells were stained with TCR{gamma}{delta}-FITC, CXCR4-PE, or CCR5-PE, CD3-TC, and CD4-allophycocyanin. Dot plots were drawn as follows: TCR{gamma}{delta}-FITC vs CD3-TC (A and D), CD4-allophycocyanin vs CXCR4-PE on the TCR{gamma}{delta}+ gated population (B and E) and CD4-allophycocyanin vs CCR5-PE on the TCR{gamma}{delta}+ population (C and F).

 
TCR{gamma}{delta}+ cells are productively infected by X4 and R5 HIV-1

To investigate productive infection of TCR{gamma}{delta}+ cells by HIV-1 in vivo, we used the SCID-hu mouse model that supports human thymopoiesis in the thy/liv graft. Seven grafted mice were infected with the X4 molecular clone (NL4-3) and seven were mock infected with control supernatant. Five weeks postinfection with NL4-3, combined multiparameter cell surface immunophenotyping and intracellular staining for HIV-1 Gag protein expression was performed on thymocyte cell suspensions. At 5 wk postinfection with X4 virus, severe depletion, as defined by <25% CD4+CD8+ double-positive cells, had occurred in three of the seven mice and intermediate levels of depletion (30–60% double positive) in the remaining four mice. As viral replication continued, loss of CD4+ cells in the total population accompanied the loss of CD4+ TCR{gamma}{delta} cells (Fig. 2Go). The percentage of TCR{gamma}{delta}+ cells expressing X4 virus ranged from 1.8 to 4.9% (mean, 3.0 ± 1.2%; Fig. 3Go). Although these levels appear quite low in proportion, particularly in the small TCR{gamma}{delta} thymocyte population, they are consistent in the seven mice tested and are of comparable frequency to the HIV-1 expression in the total thymocyte population, 0.4–8% (mean, 3.4 ± 3.0%).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. CD4 expression levels in TCR{gamma}{delta}+ thymocytes. Thymocytes were obtained from several transplant series of mock-infected and HIV-infected SCID-hu mice at 5 wk (NL4-3) and 7–9 wk (JR-CSF) postinfection and immunophenotyped with TCR{gamma}{delta}- and CD4-conjugated Abs. The mean level (solid line) of CD4 expression on the TCR{gamma}{delta}+ cells was determined (39% mock, n = 13; 13% NL4-3, n = 7; 49% JR-CSF series 1 and 2, n = 9; and 10% JR-CSF series 3, n = 4).

 


View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 3. X4 and R5 HIV-1 expression in thymic TCR{gamma}{delta}+ cells. Thymic transplants of SCID-hu mice were infected with X4-HIV-1 (NL4-3), R5-HIV-1 (JR-CSF), or mock infected with the appropriate control supernatants. Tissues were collected at either 5 wk postinfection for mock (A and B)- and NL4-3 (C and D)-infected implants or 7 wk postinfection for JR-CSF (E and F)-infected implants. Four-color intracellular flow cytometric analysis was used to identify the phenotype of virus-expressing cells. Combined surface and intracellular staining of thymocytes was performed as follows: TCR{gamma}{delta}-FITC and CD3-allophycocyanin stained the surface of the cell (A, C, and E) and KC57-PE stained the permeablized thymocytes for intracellular HIV-1 gag protein (B, D, and F). The number of events for each dot plot is as follows: A, 56,000; B, 59; C, 56,000; D, 163; E, 204,000; and F, 560.

 
The R5 virus JR-CSF was used to determine whether TCR{gamma}{delta}+ thymocytes are productively infected in vivo via CCR5. Thirteen mice were infected with JR-CSF and six were mock infected with control supernatant in three separate transplant series. In the first two series, tissue was collected at 7 and 9 wk after infection when little depletion of CD4-bearing cells was observed. Back gating on TCR{gamma}{delta}+ thymocytes for HIV-1 Gag expression in these series revealed that 2.1–9.6% (mean, 4.9 ± 2.5%) of TCR{gamma}{delta}+ thymocytes expressed virus (Fig. 3Go). Consistent with the relative lack of virus-mediated depletion of the CD4+CD8+ thymocytes in these transplant series, the level of CD4 intensity and the percent CD4+ cells was unchanged in mock- and virus-infected samples (Fig. 2Go). However, the frequency of R5 virus-expressing TCR{gamma}{delta}+ cells (4.9 ± 2.5%) exceeded the frequency of virus expression in the total population (0.99 ± 0.36%), consistent with the greater proportion of CCR5+ cells in the TCR{gamma}{delta}+ compartment than the total thymocyte population. In contrast to the first two JR-CSF-infected series, in the third transplant series infected with JR-CSF, collected at 8 wk after infection, there were moderate to severe levels of depletion of CD4+CD8+ thymocytes in the total population. As with X4 virus infection, a depletion of CD4+ TCR{gamma}{delta} cells was observed coincident with CD4 cell depletion in the total population by R5 HIV-1 in this series (Fig. 2Go).

Despite the higher frequency of CXCR4+TCR{gamma}{delta}+ cells than CCR5+TCR{gamma}{delta}+ cells in the thymus, a greater percentage of TCR{gamma}{delta}+ cells expressed virus after infection with R5 than X4 virus. However, the high frequency of R5 HIV-1 expression in the TCR{gamma}{delta}+ population only occurred in the first two transplant series infected with R5 HIV-1 when little or no CD4 cell depletion occurred in either the TCR{gamma}{delta}+ or total population. In the case of R5 HIV-1-mediated CD4 cell depletion (third transplant series), the proportion of virus expression in the TCR{gamma}{delta}+ population (0.7–2.4%) was markedly lower than that of the first two series (4.9 ± 2.5%). Therefore, the extent of HIV-1 infection in the TCR{gamma}{delta}+ population is likely due to not only the number of target cells within the population but the cytopathic effects of the virus as well. Fig. 3Go also shows an apparent enrichment of TCR{gamma}{delta} thymocytes in HIV-1-infected samples compared with the mock-infected tissues; however, these values fall within the normal range of frequencies of TCR{gamma}{delta} cells (0.22 ± 0.16%) in 17 mock-infected tissues tested.

TCR{gamma}{delta}+ thymocytes can be productively infected in vitro

To further verify HIV-1 infection of the rare TCR{gamma}{delta}+ thymocyte population, we exposed the TCR{gamma}{delta} thymocytes to X4 HIV-1 NL4-3 in vitro. TCR{gamma}{delta} thymocytes from uninfected pediatric patients were magnetically purified, infected in vitro, and cultured in serum-free medium supplemented with IL-4 to increase the levels of TCR{gamma}{delta} thymocytes as we previously reported (50). We found that TCR{gamma}{delta}+ thymocytes proliferated 3-fold in the presence of IL-4, from 2 x 105 cells at the start of culture to 6 x 105 cells after 7 days, and produced increasing amounts of viral p24 in the culture supernatant (Fig. 4Go). Viral replication in the TCR{gamma}{delta} population was further verified by intracellular Gag staining by flow cytometry (data not shown). In contrast to the TCR{gamma}{delta} populations, the unfractionated thymocyte population and the TCR{gamma}{delta}-depleted population did not expand in culture with IL-4 and failed to produce detectable levels of viral p24 (Fig. 4Go).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 4. HIV-1 replication in TCR{gamma}{delta}+ thymocytes in vitro. MACS for thymic TCR{gamma}{delta} cells enriched the population to 75% purity. The TCR{gamma}{delta}+ (positively selected), TCR{gamma}{delta}- (negatively selected), and total thymocyte populations were infected with X4 NL4-3 at a multiplicity of infection of 0.035 and were cultured for 7 days in serum-free medium containing IL-4 (20 ng/ml). A, On days 1, 4, 6, and 7 postinfection, 50 µl of supernatant was collected for ELISA measurement of viral p24. B, At day 7 postinfection, the cells in culture were counted.

 
Canonical NK T cells are undetectable in the human thymus

NKRP1A (or CD161) serves a costimulatory role on the canonical immunoregulatory NK T cells with the invariant TCR (V{alpha}24/J{alpha}Q) (35). Although murine data show the presence of the invariant TCR-possessing NK T counterpart in the murine thymus (51) and that development occurs by selection on CD1d stromal cells (52), we were unable to identify the V{alpha}24/CD161+ T cells in the human thymus by multiparameter flow cytometric analysis. Furthermore, using a mAb, 6B11, raised to the CDR3 junctional region of the V{alpha}24/J{alpha}Q, we consistently found 6B11+ cells in the peripheral blood expressing the appropriate markers of V{alpha}24 and CD161 (Fig. 5Go). However, in the thymus, the small proportion of apparent 6B11+ cells did not express the identifying NK T markers of V{alpha}24 and CD161. In addition, using CD1d tetramers loaded with the artificial ligand for canonical NK T cells, {alpha}-galactosylceramide molecule derived from a marine sponge, we were able to identify these CD1d tetramer-reactive invariant NK T cells in the periphery but not in the thymus (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 5. Canonical V{alpha}24/CD161+ NK T cells are found in the peripheral blood but not in the thymus. Four-color flow cytometric analysis of PBMC electronically gated on the lymphocytes by forward and side scatter (A–C) and thymocytes (D–F). Cells were stained with 6B11-FITC, V{alpha}24-PE, or CD161-PE, CD4-TC, or CD69-TC, and CD3-allophycocyanin. Dot plots were drawn on 6B11-FITC vs CD3-allophycocyanin (A and D, respectively) for electronic gating on 6B11+ NK T cells. The gated NK T cells were displayed for CD4-TC vs V{alpha}24-PE (B and E, respectively) and CD69-TC vs CD161-PE (C and F, respectively).

 
In the event that the NK T cells in the thymus may not be prevalent enough or are present in an immature stage as recently described for murine NK T cells (53, 54), we sought to enrich for them by culturing thymocytes with the NK-stimulating cytokines IL-2 and IL-15 (55, 56). After 7 days of culture of normal human postnatal thymocytes with IL-2 and IL-15 (20 ng/ml), both CD161 and CD56 expression on CD3+ thymocytes and CD56+/CD161+ conventional NK cells (CD3-) were increased (Fig. 6Go). Yet, there was still no detectable level of V{alpha}24+6B11+ canonical NK T cells in the stimulated population. Thus, by several methods we were unable to identify canonical NK T cells in the thymus, despite consistent detection in the peripheral blood.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 6. IL-2- and IL-15-stimulated NK T thymocytes. Human thymocytes were cultured in medium alone (A and B) or medium supplemented with IL-2 (20 ng/ml) and IL-15 (20 ng/ml; C–E). After 7 days of culture, cells were immunophenotyped and dot plots were drawn on the total population as follows: CD161-FITC vs CD3-allophycocyanin (A and C) and 6B11-FITC vs V{alpha}24-PE (B and D). A histogram was drawn on the CD161+CD3+ gated population shown in C for CD56-PE expression (E).

 
CD161+ T cells express CD4 and coreceptors for HIV-1

In lieu of the canonical NK T cells, we studied the CD161-expressing CD3+ thymocytes. As reported by others, we found the type II C-type lectin-binding protein NKRP1 (or CD161) expressed on NK cells and a small proportion of T cells in the peripheral blood (32). In the thymus, CD161 is expressed on 0.1–0.7% of the CD3+ thymocytes (Fig. 6Go). Backgating on the CD161+CD3+ thymocytes expanded with the IL-2 and IL-15 revealed an overlap with CD56-expressing CD3+ thymocytes, suggesting the existence of different types of thymocytes bearing NK markers that may represent functionally distinct subpopulations (Fig. 6Go). Furthermore, the proportion of CD4 expression on CD161+ T cells in the thymus showed a statistically significant difference (p = 0.000018) between fetal and postnatal thymocytes, 20–55% and 50–80%, respectively (Fig. 7Go), suggesting the potential for age-related variations in the development of the CD161+CD3+ thymocytes as well.



View larger version (9K):
[in this window]
[in a new window]
  		FIGURE 7. CD4 expression levels on CD161+CD3+ thymocytes. Fetal and postnatal thymocytes were immunophenotyped with CD3-, CD161-, and CD4-conjugated Abs and analyzed by multiparameter flow cytometric analysis. The mean level of CD4 expression (solid line) on the fetal thymocytes was 34 (n = 7) and postnatal thymocytes was 68 (n = 15).

 
All CD4+CD161+ T cells in peripheral blood and thymus expressed CXCR4 (Fig. 8Go). In contrast, CCR5 was expressed on ~50% of CD4+CD161+ T cells in the periphery and 20% of CD4+CD161+ T cells in the thymus (Fig. 8Go). Thus, there are more potential targets for an X4 HIV-1 than R5 HIV-1 in the CD161+ T cell population in both the periphery and thymus.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 8. CD4, CCR5, and CXCR4 on CD161+ T cells. Four-color flow cytometric analysis of fetal thymocytes (A–C) and PBMC gated on the lymphocyte gate (D–F). Cells were stained with CD161-FITC, CXCR4-PE, or CCR5-PE, CD4-TC, and CD3-allophycocyanin. Dot plots were drawn as follows: CD161-FITC vs CD3-allophycocyanin (A and D, respectively), CD4-TC vs CXCR4-PE on the CD3+CD161+ gated population (B and E, respectively), and CD4-TC vs CCR5-PE on the CD3+CD161+ gated population (C and F, respectively).

 
CD161+CD3+ thymocytes are less susceptible to productive HIV-1 infection than the general thymocyte population

As with conventional thymocytes, HIV-1 infection leads to the loss of CD4-bearing CD161+ thymocytes and enrichment of the CD4-CD161+ thymocytes reached as much as 100-fold over mock-infected thymic implants (Fig. 9Go). Furthermore, the CD56-CD161+CD3+ thymocytes were selectively increased over the CD56+CD161+CD3+ thymocytes as described in the in vitro culture with IL-2 and IL-15. More than 80% of these CD4-CD161+ thymocytes were CD8 single positive (data not shown). We previously showed HIV-1 expression in mature CD4-CD3+ conventional thymocytes (CD8+ and CD4-CD8-), which was due to infection at a previous CD4+ stage (57, 58). However, the CD4-CD161+ thymocytes were conspicuously deficient in producing HIV-1 (Fig. 9Go). In the implant with the highest level of CD3+CD161+ thymocyte enrichment, CD4-CD161+ and CD4- conventional thymocytes reached nearly equal proportions at 44 and 47%, respectively. Yet, only 0.34% of the CD4-CD161+ thymocytes expressed virus as compared with 9.2% of the conventional CD4- thymocytes, a 27-fold difference in the frequency of virus-expressing cells (Fig. 9Go). Thus, CD8+ single-positive CD161+ thymocytes could potentially develop without prior CD4 expression.



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 9. Lower X4 HIV-1 expression in CD161+ T cells than in conventional T cells. SCID-hu mice were infected by X4-HIV-1 (NL4-3) or were sham infected with the control supernatants in the human thymic implants. Thymus tissues were collected at 5 wk postinfection for mock (A and B)- and NL4-3 (C–E)-infected implants. Combined surface and intracellular staining of thymocytes was performed as follows: CD161-PE or CD4-PE, CD4-TC or CD8-TC, CD3-allophycocyanin stained the surface of the cell, and KC57-FITC stained intracellularly. Dot plots were drawn on the total population as follows: CD8-TC vs CD4-PE (A and C, respectively) and CD4-TC vs CD161-PE (B and D, respectively). Lastly, a dot plot was drawn on the CD3+ virus-expressing cells (gate not shown) and displayed as CD4-TC vs CD161-PE (E).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we examined whether specialized T cells in the thymus, namely, TCR{gamma}{delta}+ and CD161+ T cells, can be disrupted directly or indirectly by infection with HIV-1. To what extent these specialized T cells in the periphery are derived from the thymus is not fully known. Nevertheless, understanding how they respond to both X4 and R5 HIV-1 in the thymus would not only provide a better insight to HIV-1 pathogenesis in the thymus but would also have implications for HIV-1-induced perturbations of these cells in the periphery.

We describe for the first time that TCR{gamma}{delta}+ cells in the thymus are productively infected in vivo, in the SCID-hu mouse, by both R5 and X4 HIV-1. Others have reported infection of thymic TCR{gamma}{delta} clones in vitro (59, 60), suggesting the potential for direct infection by HIV-1 in vivo. For the most part, TCR{gamma}{delta} cells in the periphery do not express CD4, and thus the only point at which TCR{gamma}{delta}+ cells are naturally susceptible to HIV-1 infection is during development in the thymus when CD4 is expressed (23). We showed that both thymic and peripheral TCR{gamma}{delta}+ cells express CXCR4 and to a lesser extent CCR5, suggesting that the limiting factor for HIV-1 infection of peripheral TCR{gamma}{delta}+ cells is the expression of CD4.

As described with TCR{alpha}{beta}+ cells, CD4-bearing TCR{gamma}{delta}+ cells decline coincident with the total CD4 thymocyte loss after X4 HIV-1 (NL4-3) infection. Although it appears in Fig. 3Go that HIV infection leads to an enrichment of TCR{gamma}{delta} cells by both R5 and X4 HIV, we were unable to determine a significant level of enrichment. The frequencies of TCR{gamma}{delta} cells in the HIV-infected implants (0.3 and 0.27%) are within the range of the frequencies of TCR{gamma}{delta} thymocytes in 17 noninfected thy/liv implants (0.22 ± 0.16%).

As for functional responsiveness of TCR{gamma}{delta}+ cells after HIV-1 infection, in TCR{gamma}{delta}+ cell clones, HIV-1 infection reportedly does not affect cytokine gene expression or proliferative response to Daudi cells in vitro (60). We also report here that TCR{gamma}{delta}+ cells proliferate in response to IL-4 after in vitro HIV-1 infection. However, in ex vivo assays V{gamma}9/V{delta}2 in HIV-1-infected individuals are anergic to phosphoantigen stimulation from HIV-1-infected individuals as compared with normal healthy individuals (61). Therefore, functional changes of TCR{gamma}{delta}+ cells may be present in vivo and may play an important role in immunodeficiency in HIV-1 infection.

HIV-1 infection of TCR{gamma}{delta}+ cells in the thymus may contribute to viral spread as these TCR{gamma}{delta}+ cells migrate to peripheral sites. The predominant TCR{gamma}{delta}+ cell subset in the thymus is the V{delta}1+ T cell subset, which is also the dominant in the spleen and the major T cell population in the intestinal epithelia (15). Furthermore, V{delta}1+TCR{gamma}{delta}+ cells are reported in other peripheral sites such as the nasal mucosa (62), oral epithelia (63), lungs (64), semen (65), and decidua of the placenta during pregnancy (66). Localization of V{delta}1 TCR{gamma}{delta}+ cells in some of these peripheral sites is thought to be by a integrin-mediated extravasation capability unique to V{delta}1+ T cells (67). Although it has been described that TCR{gamma}{delta} development takes place in extrathymic sites during fetal development, such as the liver and primitive gut as early as 6 wk of gestation preceding the formation of the thymic rudiment (68, 69, 70), determination of extrathymic development of human TCR{gamma}{delta}+ cells with a functional thymus has proven enigmatic (71). Therefore, HIV-1 infection of TCR{gamma}{delta}+ cells during thymopoiesis has implications for HIV-1 pathogenesis in epithelial and mucosal sites, particularly in highly active postnatal thymus of pediatric patients.

We show here that based on flow cytometric analysis we were unable to identify the invariant V{alpha}24/J{alpha}Q TCR and CD161+ NK T cells in the human thymus despite consistent detection in the peripheral blood. Data in the mouse show both thymic and extrathymic development of the murine counterpart (V{alpha}14/J{alpha}281 NK1.1+ T cells) based on thymic grafts in athymic nude mice (51) and bone marrow reconstitution studies in adult thymectomized and nude irradiated mice (72), respectively. Whether the human canonical V{alpha}24 invariant NK T cells in the peripheral blood are extrathymic derived or thymic derived remains to be determined.

T cells expressing the NKRP1A (CD161) with a diverse repertoire are found in the thymus at ~0.3% of the total thymocyte population. We show that X4-HIV-1 infection of the thymus leads to an enrichment of CD161+ T cells; however, the enrichment of these cells is disproportionate between CD4+ and CD4-CD161+ T cells. As with HIV-1-mediated loss of CD4-bearing thymocytes either by direct or indirect effects of HIV-1, CD4+CD161+ thymocytes are likewise decreased, presumably by direct infection since virus-expressing cells are detected in the few CD4+CD161+ T cells present. However, unlike the CD4- T cells in the thymus that express virus after depletion of CD4+CD8+ thymocytes, CD4-CD161+ thymocytes contain as much as 27-fold less virus-expressing cells. It has been previously shown that the mechanism of virus expression in CD4+ thymocytes is due to infection at an earlier CD4-bearing stage (58, 73). CD4-CD161+ thymocytes may therefore not pass through a CD4+ stage during development, thereby limiting access of HIV-1 and explaining the paucity of virus-expressing cells in this population.

It is known that CD161+ T cells are found in the liver. The SCID-hu fetal thy/liv implant comprises fetal liver components as a source of hemopoietic stem cells. Therefore, we investigated the proportions and phenotype of CD161+ thymocytes in fetal and postnatal thymus specimens as compared with cells derived from the SCID-hu thy/liv implant. There was no difference in the percentages of CD161+ or CD4+ and CD8+ thymocytes in fetal thymus and SCID-hu thy/liv thymocytes, thereby discounting any potential contribution of CD161+ T cells by liver components in the thymus graft. However, there was a statistically significant difference in the percentages of CD4+CD161+ cells between fetal and postnatal thymocytes. These differences could be due to waves of CD161+ T cell development at the time of birth as reported for the general thymocyte population (74) or due to the source of the hemopoietic precursors, liver (fetal life) vs the bone marrow (postnatal life).

Expression of a NK-associated marker, CD161, on T cells may exhibit unique functional activity and/or regulatory control. CD161 has been reported to function as a costimulatory molecule on the invariant V{alpha}24 NK T cells for proliferation and cytokine secretion (35) and as a negative regulator of cytotoxicity of NK cells (32). However, the role of CD161 expression in chronically activated CD8 T cytotoxic cells in the mouse (75) and humans (36, 76, 77) has not been elucidated. Nevertheless, given the expression on effector/senescent T cells, which display decreased functional activity (37), it likely functions to negatively influence cytotoxic activity.

The increased frequency of CD161+ T cells in the HIV-infected thymus is evident after HIV-induced depletion of the numerous CD4 target cells in the thymus. How they arise is another question. Endogenous CD161+CD3+ thymocytes could be merely enriched in number as the numerous CD4 target cells are depleted. Alternatively, HIV replication could result in chronic activation of a subset of CD8 T cells inducing CD161 upon reaching senescence. Further work is necessary for determining the function of this population in the thymus.

We have shown that minor, but important cell populations in the thymus are targets for HIV infection and express HIV-1. Therefore, given the significant roles TCR{gamma}{delta} and NK T cells play in both innate and acquired immunity, HIV-1 infection and/or disruption of these cells may contribute to the overall immune dysfunction seen in HIV-1 disease.


   Acknowledgments
 
We thank Hillel Laks and his colleagues and staff for providing us with the thymus specimens and Deborah Anisman-Posner and Silvia Neagos for their excellent technical assistance.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (HD 29341, HD 37597, AI 32440, University of California, Los Angeles Center for AIDS Research). Back

2 Address correspondence and reprint requests to Dr. Christel H. Uittenbogaart, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1747. E-mail address: uittenbo{at}ucla.edu Back

3 Abbreviations used in this paper: NKRP1, NK receptor protein 1; TC, Tricolor; thy/liv, thymus/liver; i.u., infectious units. Back

Received for publication May 23, 2002. Accepted for publication August 27, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burke, A. P., D. Anderson, W. Benson, R. Turnicky, P. Mannan, Y. H. Liang, J. Smialek, R. Virmani. 1995. Localization of human immunodeficiency virus 1 RNA in thymic tissues from asymptomatic drug addicts. Arch. Pathol. Lab. Med 119:36.[Medline]
  2. Prevot, S., J. Audouin, J. Andre-Bougaran, R. Griffais, A. Le Tourneau, J. G. Fournier, J. Diebold. 1992. Thymic pseudotumorous enlargement due to follicular hyperplasia in a human immunodeficiency virus sero-positive patient. Am. J. Clin. Pathol. 97:420.[Medline]
  3. Jamieson, B. D., J. A. Zack. 1999. Murine models for HIV disease. AIDS 13:(Suppl. A):S5.
  4. Joshi, V. V., J. M. Oleske. 1985. Pathologic appraisal of the thymus gland of acquired immunodeficiency syndrome in children: a study of four cases and review of the literature. Arch. Pathol. Lab. Med. 109:142.[Medline]
  5. Joshi, V. V., J. M. Oleske, S. Saad, C. Gadol, E. Connor, R. Bobila, A. B. Minnefor. 1986. Thymus biopsy in children with acquired immunodeficiency syndrome. Arch. Pathol. Lab. Med. 110:837.[Medline]
  6. McCune, J. M., R. Namikawa, H. Kaneshima, L. D. Shultz, M. Lieberman, I. L. Weissman. 1988. The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241:1632.[Abstract/Free Full Text]
  7. Wykrzykowska, J. J., M. Rosenzweig, R. S. Veazey, M. A. Simon, K. Halvorsen, R. C. Desrosiers, R. P. Johnson, A. A. Lackner. 1998. Early regeneration of thymic progenitors in rhesus macaques infected with simian immunodeficiency virus. J. Exp. Med. 187:1767.[Abstract/Free Full Text]
  8. Pedroza-Martins, L., K. B. Gurney, B. E. Torbett, C. H. Uittenbogaart. 1998. Differential tropism and replication kinetics of human immunodeficiency virus type 1 isolates in thymocytes: coreceptor expression allows viral entry, but productive infection of distinct subsets is determined at the postentry level. J. Virol. 72:9441.[Abstract/Free Full Text]
  9. Jamieson, B. D., S. Pang, G. M. Aldrovandi, J. Zha, J. A. Zack. 1995. In vivo pathogenic properties of two clonal HIV-1 isolates. J. Virol. 69:6259.[Abstract]
  10. Berkowitz, R. D., K. P. Beckerman, T. J. Schall, J. M. McCune. 1998. CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation. J. Immunol. 161:3702.[Abstract/Free Full Text]
  11. Kaneshima, H., L. Su, M. L. Bonyhadi, R. I. Connor, D. D. Ho, J. M. McCune. 1994. Rapid-high, syncytium-inducing isolates of human immunodeficiency virus type 1 induce cytopathicity in the human thymus of the SCID-hu mouse. J. Virol. 68:8188.[Abstract/Free Full Text]
  12. Groh, V., A. Steinle, S. Bauer, T. Spies. 1998. Recognition of stress-induced MHC molecules by intestinal epithelial {gamma}{delta} T cells. Science 279:1737.[Abstract/Free Full Text]
  13. Spada, F. M., E. P. Grant, P. J. Peters, M. Sugita, A. Melian, D. S. Leslie, H. K. Lee, E. van Donselaar, D. A. Hanson, A. M. Krensky, et al 2000. Self-recognition of CD1 by {gamma}/{delta} T cells: implications for innate immunity. J. Exp. Med. 191:937.[Abstract/Free Full Text]
  14. Davodeau, F., M. A. Peyrat, A. Necker, R. Dominici, F. Blanchard, C. Leget, J. Gaschet, P. Costa, Y. Jacques, A. Godard, et al 1997. Close phenotypic and functional similarities between human and murine {alpha}{beta} T cells expressing invariant TCR {alpha}-chains. J. Immunol. 158:5603.[Abstract]
  15. Porcelli, S., M. B. Brenner, H. Band. 1991. Biology of the human gamma delta T-cell receptor. Immunol. Rev. 120:137.[Medline]
  16. Tanaka, Y., S. Sano, E. Nieves, G. De Libero, D. Rosa, R. L. Modlin, M. B. Brenner, B. R. Bloom, C. T. Morita. 1994. Nonpeptide ligands for human {gamma}{delta} T cells. Proc. Natl. Acad. Sci. USA 91:8175.[Abstract/Free Full Text]
  17. Tanaka, Y., C. T. Morita, E. Nieves, M. B. Brenner, B. R. Bloom. 1995. Natural and synthetic non-peptide antigens recognized by human {gamma}{delta} T cells. Nature 375:155.[Medline]
  18. Parker, C. M., V. Groh, H. Band, S. A. Porcelli, C. Morita, M. Fabbi, D. Glass, J. L. Strominger, M. B. Brenner. 1990. Evidence for extrathymic changes in the T cell receptor {gamma}/{delta} repertoire. J. Exp. Med. 171:1597.[Abstract/Free Full Text]
  19. Casorati, G., G. De Libero, A. Lanzavecchia, N. Migone. 1989. Molecular analysis of human {gamma}/{delta}+ clones from thymus and peripheral blood. J. Exp. Med. 170:1521.[Abstract/Free Full Text]
  20. Rossol, R., J. M. Dobmeyer, T. S. Dobmeyer, S. A. Klein, S. Rossol, D. Wesch, D. Hoelzer, D. Kabelitz, E. B. Helm. 1998. Increase in V{delta}1+ {gamma}{delta} T cells in the peripheral blood and bone marrow as a selective feature of HIV-1 but not other virus infections. Br. J. Haematol. 100:728.[Medline]
  21. Wesch, D., T. Hinz, D. Kabelitz. 1998. Analysis of the TCR V{gamma} repertoire in healthy donors and HIV-1-infected individuals. Int. Immunol. 10:1067.[Abstract/Free Full Text]
  22. Spits, H., X. Paliard, Y. Vandekerckhove, P. van Vlasselaer, J. E. de Vries. 1991. Functional and phenotypic differences between CD4+ and CD4- T cell receptor-{gamma}{delta} clones from peripheral blood. J. Immunol. 147:1180.[Abstract]
  23. Offner, F., K. Van Beneden, V. Debacker, D. Vanhecke, B. Vandekerckhove, J. Plum, G. Leclercq. 1997. Phenotypic and functional maturation of TCR {gamma}{delta} cells in the human thymus. J. Immunol. 158:4634.[Abstract]
  24. Takahashi, T., M. Nieda, Y. Koezuka, A. Nicol, S. A. Porcelli, Y. Ishikawa, K. Tadokoro, H. Hirai, T. Juji. 2000. Analysis of human V{alpha}24+CD4+ NKT cells activated by {alpha}-glycosylceramide-pulsed monocyte-derived dendritic cells. J. Immunol. 164:4458.[Abstract/Free Full Text]
  25. Takahashi, T., S. Chiba, M. Nieda, T. Azuma, S. Ishihara, Y. Shibata, T. Juji, H. Hirai. 2002. Cutting edge: analysis of human V{alpha}24+CD8+ NK T cells activated by {alpha}-galactosylceramide-pulsed monocyte-derived dendritic cells. J. Immunol. 168:3140.[Abstract/Free Full Text]
  26. Yang, O. O., F. K. Racke, P. T. Nguyen, R. Gausling, M. E. Severino, H. F. Horton, M. C. Byrne, J. L. Strominger, S. B. Wilson. 2000. CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolytic activity of V{alpha}24J{alpha}Q T cells: a feedback mechanism for immune regulation. J. Immunol. 165:3756.[Abstract/Free Full Text]
  27. D’Andrea, A., D. Goux, C. De Lalla, Y. Koezuka, D. Montagna, A. Moretta, P. Dellabona, G. Casorati, S. Abrignani. 2000. Neonatal invariant V{alpha}24+ NKT lymphocytes are activated memory cells. Eur. J. Immunol. 30:1544.[Medline]
  28. Kadowaki, N., S. Antonenko, S. Ho, M. C. Rissoan, V. Soumelis, S. A. Porcelli, L. L. Lanier, Y. J. Liu. 2001. Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells. J. Exp. Med. 193:1221.[Abstract/Free Full Text]
  29. Godfrey, D. I., K. J. Hammond, L. D. Poulton, M. J. Smyth, A. G. Baxter. 2000. NKT cells: facts, functions and fallacies. Immunol. Today 21:573.[Medline]
  30. van der Vliet, H. J., B. M. von Blomberg, M. D. Hazenberg, N. Nishi, S. A. Otto, B. H. van Benthem, M. Prins, F. A. Claessen, A. J. van den Eertwegh, G. Giaccone, et al 2002. Selective decrease in circulating V{alpha}24+V{beta}11+ NKT cells during HIV type 1 infection. J. Immunol. 168:1490.[Abstract/Free Full Text]
  31. Exley, M. A., S. M. Tahir, O. Cheng, A. Shaulov, R. Joyce, D. Avigan, R. Sackstein, S. P. Balk. 2001. A major fraction of human bone marrow lymphocytes are Th2-like CD1d-reactive T cells that can suppress mixed lymphocyte responses. J. Immunol. 167:5531.[Abstract/Free Full Text]
  32. Lanier, L. L., C. Chang, J. H. Phillips. 1994. Human NKR-P1A: a disulfide-linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes. J. Immunol. 153:2417.[Abstract]
  33. Exley, M., J. Garcia, S. P. Balk, S. Porcelli. 1997. Requirements for CD1d recognition by human invariant V{alpha}24+CD4-CD8- T cells. J. Exp. Med. 186:109.[Abstract/Free Full Text]
  34. McMahon, C. W., D. H. Raulet. 2001. Expression and function of NK cell receptors in CD8+ T cells. Curr. Opin. Immunol. 13:465.[Medline]
  35. Exley, M., S. Porcelli, M. Furman, J. Garcia, S. Balk. 1998. CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant V{alpha}24J{alpha}Q T cell receptor {alpha} chains. J. Exp. Med. 188:867.[Abstract/Free Full Text]
  36. Tarazona, R., O. DelaRosa, J. G. Casado, J. Torre-Cisneros, J. L. Villanueva, M. D. Galiani, J. Pena, R. Solana. 2002. NK-associated receptors on CD8 T cells from treatment-naive HIV-infected individuals: defective expression of CD56. AIDS 16:197.[Medline]
  37. Tarazona, R., O. DelaRosa, C. Alonso, B. Ostos, J. Espejo, J. Pena, R. Solana. 2000. Increased expression of NK cell markers on T lymphocytes in aging and chronic activation of the immune system reflects the accumulation of effector/senescent T cells. Mech. Ageing Dev. 121:77.[Medline]
  38. Uittenbogaart, C. H., D. J. Anisman, J. A. Zack, A. Economides, I. Schmid, E. F. Hays. 1995. Effects of cytokines on HIV-1 production by thymocytes. Thymus 23:155.
  39. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284.[Abstract/Free Full Text]
  40. Koyanagi, Y., S. Miles, R. T. Mitsuyasu, J. E. Merill, H. V. Vinters, I. S. Y. Chen. 1987. Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 236:819.[Abstract/Free Full Text]
  41. Ferbas, J., A. H. Kaplan, M. A. Hausner, L. E. Hultin, J. L. Matud, Z. Liu, D. L. Panicali, H. Nerng-Ho, R. Detels, J. V. Giorgi. 1995. Virus burden in long-term survivors of human immunodeficiency virus (HIV) infection is a determinant of anti-HIV CD8+ lymphocyte activity. J. Infect. Dis. 172:329.[Medline]
  42. Zack, J. A., S. J. Arrigo, S. R. Weitman, A. S. Go, A. Haislip, I. S. Y. Chen. 1990. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61:213.[Medline]
  43. Zack, J. A., A. M. Haislip, P. Krogstad, I. S. Y. Chen. 1992. Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle. J. Virol. 66:1717.[Abstract/Free Full Text]
  44. Aldrovandi, G. M., G. Feuer, L. Gao, B. Jamieson, M. Kristeva, I. S. Y. Chen, J. A. Zack. 1993. The SCID-hu mouse as a model for HIV-1 infection. Nature 363:732.[Medline]
  45. Bonyhadi, M. L., L. Rabin, S. Salimi, D. A. Brown, J. Kosek, J. M. McCune, H. Kaneshima. 1993. HIV induces thymus depletion in vivo. Nature 363:728.[Medline]
  46. Stanley, S. K., J. M. McCune, H. Kaneshima, J. S. Justement, M. Sullivan, E. Boone, M. Baseler, J. Adelsberger, M. Bonyhadi, J. Orenstein, et al 1993. Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse. J. Exp. Med. 178:1151.[Abstract/Free Full Text]
  47. Jamieson, B. D., G. M. Aldrovandi, V. Planelles, J. B. M. Jowett, L. Gao, L. Bloch, I. S. Y. Chen, J. A. Zack. 1994. Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity. J. Virol. 68:3478.[Abstract/Free Full Text]
  48. Schmid, I., C. H. Uittenbogaart, J. V. Giorgi. 1991. A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry 12:279.[Medline]
  49. Schmid, I., W. J. Krall, C. H. Uittenbogaart, J. Braun, J. V. Giorgi. 1992. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13:204.[Medline]
  50. Uittenbogaart, C. H., S. Higashitani, I. Schmid, L. W. Vollger, T. Boone, L. T. Clement. 1990. Interleukin-4 induces expression of the CD45RA antigen on human thymocyte subpopulations. Int. Immunol. 2:1179.[Abstract/Free Full Text]
  51. Levitsky, H. I., P. T. Golumbek, D. M. Pardoll. 1991. The fate of CD4-8- T cell receptor-{alpha}{beta}+ thymocytes. J. Immunol. 146:1113.[Abstract]
  52. Gapin, L., J. L. Matsuda, C. D. Surh, M. Kronenberg. 2001. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nat. Immunol. 2:971.[Medline]
  53. Benlagha, K., T. Kyin, A. Beavis, L. Teyton, A. Bendelac. 2002. A thymic precursor to the NK T cell lineage. Science 296:553.[Abstract/Free Full Text]
  54. Pellicci, D. G., K. J. Hammond, A. P. Uldrich, A. G. Baxter, M. J. Smyth, D. I. Godfrey. 2002. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1-CD4+ CD1d-dependent precursor stage. J. Exp. Med. 195:835.[Abstract/Free Full Text]
  55. Uittenbogaart, C. H., I. Schmid, D. J. Anisman, S. Kiertscher, H. Shau, K. M. Zsebo, L. T. Clement. 1996. Human thymocyte responsiveness to stem cell factor: synergy with interleukin-2 for the generation of NK/LAK cytotoxicity. Immunol. Lett. 52:45.[Medline]
  56. Leclercq, G., V. Debacker, M. De Smedt, J. Plum. 1996. Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells. J. Exp. Med. 184:325.[Abstract/Free Full Text]
  57. Uittenbogaart, C. H., D. J. Anisman, B. D. Jamieson, S. Kitchen, I. Schmid, J. A. Zack, E. F. Hays. 1996. Differential tropism of HIV-1 isolates for distinct thymocyte subsets in vitro. AIDS 10:F9.[Medline]
  58. Kitchen, S. G., C. H. Uittenbogaart, J. A. Zack. 1997. Mechanism of human immunodeficiency virus type 1 localization in CD4-negative thymocytes: differentiation from a CD4-positive precursor allows productive infection. J. Virol. 71:5713.[Abstract]
  59. Schnittman, S. M., S. M. Denning, J. J. Greenhouse, J. S. Justement, M. Baseler, J. Kurtzberg, B. F. Haynes, A. S. Fauci. 1990. Evidence for susceptibility of intrathymic T-cell precursors and their progeny carrying T-cell antigen receptor phenotypes TCR{alpha}{beta}+ and TCR{gamma}/{delta}+ to human immunodeficiency virus infection: a mechanism for CD4+ (T4) lymphocyte depletion. Proc. Natl. Acad. Sci. USA 87:7727.[Abstract/Free Full Text]
  60. Wallace, M., A. M. Scharko, C. D. Pauza, P. Fisch, K. Imaoka, S. Kawabata, K. Fujihashi, H. Kiyono, Y. Tanaka, B. R. Bloom, M. Malkovsky. 1997. Functional {gamma}{delta} T-lymphocyte defect associated with human immunodeficiency virus infections. Mol. Med. 3:60.[Medline]
  61. Poccia, F., S. Boullier, H. Lecoeur, M. Cochet, Y. Poquet, V. Colizzi, J. J. Fournie, M. L. Gougeon. 1996. Peripheral V{gamma}9/V{delta}2 T cell deletion and anergy to nonpeptidic mycobacterial antigens in asymptomatic HIV-1-infected persons. J. Immunol. 157:449.[Abstract]
  62. Pawankar, R. U., M. Okuda, K. Suzuki, K. Okumura, C. Ra. 1996. Phenotypic and molecular characteristics of nasal mucosal {gamma}{delta} T cells in allergic and infectious rhinitis. Am. J. Respir. Crit. Care Med 153:1655.[Abstract]
  63. Pepin, L. F., T. Roger, J. Morisset, M. Seman. 1993. Preferential V{delta}1 expression among TCR {gamma}/{delta}-bearing T cells in human oral epithelium. Scand. J. Immunol. 37:289.[Medline]
  64. Lahn, M.. 2000. The role of {gamma}{delta} T cells in the airways. J. Mol. Med. 78:409.[Medline]
  65. Bertotto, A., F. Spinozzi, R. Gerli, G. Bassotti, G. M. Fabietti, G. Castellucci, C. Vagliasindi, R. Vaccaro. 1995. {gamma}{delta} T-cell subset distribution in human semen from fertile donors. Am. J. Reprod. Immunol. 34:176.
  66. Mincheva-Nilsson, L., M. Kling, S. Hammarstrom, O. Nagaeva, K. G. Sundqvist, M. L. Hammarstrom, V. Baranov. 1997. {gamma}{delta} T cells of human early pregnancy decidua: evidence for local proliferation, phenotypic heterogeneity, and extrathymic differentiation. J. Immunol. 159:3266.[Abstract]
  67. Cepek, K. L., S. K. Shaw, C. M. Parker, G. J. Russell, J. S. Morrow, D. L. Rimm, M. B. Brenner. 1994. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the {alpha}E 32 {beta}7 integrin. Nature 372:190.[Medline]
  68. McVay, L. D., S. R. Carding. 1996. Extrathymic origin of human {gamma}{delta} T cells during fetal development. J. Immunol. 157:2873.[Abstract]
  69. McVay, L. D., S. S. Jaswal, C. Kennedy, A. Hayday, S. R. Carding. 1998. The generation of human {gamma}{delta} T cell repertoires during fetal development. J. Immunol. 160:5851.[Abstract/Free Full Text]
  70. Krangel, M. S., H. Yssel, C. Brocklehurst, H. Spits. 1990. A distinct wave of human T cell receptor {gamma}/{delta} lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production. J. Exp. Med. 172:847.[Abstract/Free Full Text]
  71. McVay, L. D., S. R. Carding. 1999. Generation of human {gamma}{delta} T-cell repertoires. Crit. Rev. Immunol 19:431.[Medline]
  72. Kikly, K., G. Dennert. 1992. Evidence for extrathymic development of TNK cells: NK1+CD3+ cells responsible for acute marrow graft rejection are present in thymus-deficient mice. J. Immunol. 149:403.[Abstract]
  73. Lee, S., H. Goldstein, M. Baseler, J. Adelsberger, H. Golding. 1997. Human immunodeficiency virus type 1 infection of mature CD3highCD8+ thymocytes. J. Virol. 71:6671.[Abstract]
  74. Krogstad, P., C. H. Uittenbogaart, R. Dickover, Y. J. Bryson, S. Plaeger, A. Garfinkel. 1999. Primary HIV infection of infants: the effects of somatic growth on lymphocyte and virus dynamics. Clin. Immunol. 92:25.[Medline]
  75. Kambayashi, T., E. Assarsson, J. Michaelsson, P. Berglund, A. D. Diehl, B. J. Chambers, H. G. Ljunggren. 2000. Emergence of CD8+ T cells expressing NK cell receptors in influenza A virus-infected mice. J. Immunol. 165:4964.[Abstract/Free Full Text]
  76. Nickoloff, B. J., B. Bonish, B. B. Huang, S. A. Porcelli. 2000. Characterization of a T cell line bearing natural killer receptors and capable of creating psoriasis in a SCID mouse model system. J. Dermatol. Sci. 24:212.[Medline]
  77. Exley, M. A., Q. He, O. Cheng, R. J. Wang, C. P. Cheney, S. P. Balk, M. J. Koziel. 2002. Cutting edge: compartmentalization of Th1-like noninvariant CD1d-reactive T cells in hepatitis C virus-infected liver. J. Immunol. 168:1519.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CVIHome page
D. S. An, B. Poon, R. H. T. Fang, K. Weijer, B. Blom, H. Spits, I. S. Y. Chen, and C. H. Uittenbogaart
Use of a Novel Chimeric Mouse Model with a Functionally Active Human Immune System To Study Human Immunodeficiency Virus Type 1 Infection
Clin. Vaccine Immunol., April 1, 2007; 14(4): 391 - 396.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
S. Y. Thomas, C. M. Lilly, A. D. Luster, N. Pham-Thi, J. de Blic, M. C. Leite-de-Moraes, O. Akbari, J. L. Faul, and D. T. Umetsu
Invariant natural killer T cells in bronchial asthma.
N. Engl. J. Med., June 15, 2006; 354(24): 2613 - 2616.
[Full Text] [PDF]

Home page
 J. Virol.Home page
N. Ank, K. Petersen, L. Malmgaard, S. C. Mogensen, and S. R. Paludan
Age-Dependent Role for CCR5 in Antiviral Host Defense against Herpes Simplex Virus Type 2
J. Virol., August 1, 2005; 79(15): 9831 - 9841.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. K. Sandberg, C. A. Stoddart, F. Brilot, K. A. Jordan, and D. F. Nixon
Development of innate CD4+ {alpha}-chain variable gene segment 24 (V{alpha}24) natural killer T cells in the early human fetal thymus is regulated by IL-7
PNAS, May 4, 2004; 101(18): 7058 - 7063.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. A. Poles, S. Barsoum, W. Yu, J. Yu, P. Sun, J. Daly, T. He, S. Mehandru, A. Talal, M. Markowitz, et al.
Human Immunodeficiency Virus Type 1 Induces Persistent Changes in Mucosal and Blood {gamma}{delta} T Cells despite Suppressive Therapy
J. Virol., October 1, 2003; 77(19): 10456 - 10467.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gurney, K. B.
Right arrow Articles by Uittenbogaart, C. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gurney, K. B.
Right arrow Articles by Uittenbogaart, C. H.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS