International Immunology

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International Immunology, Vol. 13, No. 1, 75-83, January 2001
© 2001 Japanese Society for Immunology

CD4⁺Thy1^- thymocytes with a T_h-type 2 cytokine response

Douglas M. Cerasoli¹, Garnett Kelsoe and Marcella Sarzotti

Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 27710, USA
¹ Present address: Division of Biochemistry and Pharmacology, United States Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, APG, MD 21010-5425, USA

Correspondence to: M. Sarzotti

	Abstract

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We have identified a small subset of CD3⁺, CD4⁺, CD8^– thymocytes that do not express Thy1 (CD90). This Thy1^– subset represents 1–3.7% of the total number of thymocytes in a naive mouse. CD4⁺Thy1^– thymocytes express high levels of CD3, intermediate to high levels of heat-stable antigen (HSA), and low levels of CD25, CD45RB, CD69, CD44 and CD62L. They produce high titers of IL-4 and no IFN- upon stimulation in vitro, a response characteristic of T_h2 cells. In the thymi of mice infected neonatally with a high dose of the retrovirus Cas-Br-E MuLV, the frequency of CD4⁺Thy1^– cells increased ~10-fold. High-dose virus infection resulted in decreased HSA and increased CD44 expression on CD4⁺Thy1^– cells relative to cells from naive mice. CD4⁺Thy1^– cells from high-dose infected mice also secreted IL-4 and not IFN- upon in vitro stimulation. We previously reported that infection of newborn mice with a high dose of murine retrovirus results in the induction of a non-protective anti-viral T_h2 T cell response; CD4⁺Thy1^– thymocytes with a T_h2-like cytokine profile may play a role in determining the cytokine bias of this anti-viral response.

Keywords: Cas-Br-E MuLV, CD4⁺ cells, newborn mice, T_h2, Thy1^– thymocytes, thymic atrophy

	Introduction

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Thy1 (CD90) is a glycosylphosphatidylinositol-linked membrane protein that is expressed on mouse thymocytes, peripheral T lymphocytes, epidermal dendritic cells, hematopoietic stem cells, neural cells and fibroblasts. The function and ligand(s) of Thy1 remain undefined (1), although it is clear that the Thy1 molecule is able to transduce signals that can activate or inhibit the responses of mature T cells (2–4). There have been reports that some peripheral CD4⁺ T cell populations do not express Thy1 (Thy1^– T cells). Cells of this phenotype have been identified in mice either recovering from sub-lethal irradiation or from irradiation followed by bone marrow transplantation and in mice infected with a murine retrovirus (LP-BM5) (5–7). CD4⁺Thy1^– T cells are also localized at high relative frequency in gut-associated Peyer's patches (8) and in the germinal centers of spleen and lymph nodes (9). When tested, the CD4⁺Thy1^– T cells were shown to express ß TCR. It is unclear whether CD4⁺Thy1^– T cells are functionally distinct from other T cell populations; in T cell clones Thy1 has been shown to be transiently down-regulated in response to TCR-mediated stimulation. We now identified a small subset of CD3⁺CD4⁺CD8^– thymocytes that do not express Thy1. These CD4⁺Thy1^– thymocytes produce high titers of IL-4 and no IFN- upon stimulation in vitro, a response characteristic of T_h2 cells (10). The significance of this thymocyte subset in pathologic situations associated with T_h2 responses has not been investigated. We have previously shown that infection of newborn mice with a high dose of the retrovirus Cas-Br-E MuLV (Cas) results in the induction of a non-protective anti-viral T_h2 T cell response characterized by the production of IL-4 and no IFN- (11). Cas is an ecotropic MuLV that replicates in the spleen and the brain of neonatal susceptible mice, and induces neuropathogenic disease (12). We report here that the frequency of CD4⁺Thy1^– thymocytes is greatly enhanced in the thymi of mice infected at birth with a high dose of Cas. The potential role of these IL-4-producing, Thy1^– thymocytes in the disease process of mice infected neonatally with Cas virus is discussed.

	Methods

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Mice and viruses
Breeding pairs of NFS/NCr mice obtained from the NIH Repository Colony (Bethesda, MD) were used to establish a breeding colony in the Animal Facility of the Baltimore VA Medical Center, to provide mice for the described studies. NFS/NCr mice are Fv-1ⁿⁿ, H-2^sq4, Thy1.2⁺, NK1.1^–, express no ecotropic MuLV, and only low levels of endogenous xenotropic MuLV (13). Cas virus was grown in SC-1 fibroblast cells, titered by XC plaque assay (14) and monitored in vivo by paralysis induction (12). Mice were injected i.p. at 2 days of age with 0.03 ml of a single dose of Cas (0.3 p.f.u./mouse, i.e. low dose, or 1000 p.f.u./mouse, i.e. high dose) or with medium (control).

Antibodies and flow cytometry reagents
mAb-specific for mouse CD4 (H129.19), CD8 (53-6.7), Thy1.2 (53-2.1), B220 (RA3-6B2), CD3 (145-2C11), TCRß (H57-597), Mac-1 (M1/70), Ly-6G (Gr-1), Ly-76 (TER-119), CD25 (3C7), heat-stable antigen (HSA; M1/69), CD44 (IM7), CD45RB (16A), CD62L (MEL-14), CD69 (H1.2F3) and GL-7(GL-7) were purchased from PharMingen (San Diego, CA), and were titrated individually before use. mAb were either biotinylated or directly conjugated to FITC, phycoerythrin (PE) or allophycocyanin, depending on the combinations used in each experiment. Polyclonal goat anti-mouse gp70 antisera, obtained from the Division of Cancer Cause and Prevention (National Cancer Institute, Bethesda, MD) was detected using FITC-conjugated donkey anti-goat IgG (Dako, Carpinteria, CA). Anti-gp70 and anti-goat antisera were kindly provided by Dr D. Robbins (VAMC, Baltimore, MD). Staining with biotinylated antibodies was detected using streptavidin-conjugated allophycocyanin (Caltag, San Francisco, CA).

Flow cytometry
Single-cell suspensions of thymus cells were prepared from age-matched infected and control mice using standard methods. In some cases, mice were injected i.p. with 500 µl of a 25% solution of India ink in PBS 30 min prior to sacrifice, in order to confirm that thymic preparations did not inadvertently include cells from the parathymic lymph nodes. In all cases, cells were maintained at 4°C for the remainder of each experiment. The single-cell suspensions were depleted of red blood cells by treatment with a 0.83% solution of NH₄Cl followed by washing in PBS (pH 7.4) supplemented with 2% FCS and 0.08% sodium azide. Cells were distributed into 96-well V-bottom plates (5x10⁵ cells/well) and incubated with anti-FcRII/RIII (FcBlock; PharMingen) for 5 min. After washing, cells were labeled with biotinylated antibodies or anti-gp70 anti-sera for 30 min, washed again, and incubated with fluorochrome-conjugated antibodies, streptavidin–allophycocyanin and 7-aminoactinomyosin D (7AAD; Molecular Probes, Eugene, OR) for 30 min. After final washing, cells were analyzed on a FACSort flow cytometer (Becton Dickinson, Mountain View, CA) modified with a second laser for four-color analysis. The cytometer was calibrated and compensation levels were determined using control lymphocytes stained with single colors. Between 30,000 and 100,000 lymphocyte-sized (based on forward and side scatter parameters), viable (based on exclusion of 7AAD) cell events were saved per sample, and were analyzed using CellQuest software (version 3.01; Becton Dickinson).

MACS sorting
Single-cell suspensions of thymocytes were prepared and depleted of red blood cells as above under aseptic conditions at 4°C. After preincubation with FcBlock, cells were labeled with biotinylated antibodies specific for CD8, Thy1.2, B220, Ly-76, Ly-6G and Mac-1 for 30 min. After washing, cells were incubated with streptavidin-conjugated microbeads (Miltenyi Biotec, Gladbach, Germany) for 15 min. Microbead-labeled cells were separated from unlabeled cells by passage over a GS column (Miltenyi Biotec) in a magnetic field based on the manufacturer's protocol. Recovered, unlabeled cells were routinely 85–95% CD3⁺, CD4⁺, TCRß⁺ and Thy1.2^– based on subsequent flow cytometric analyses. In these subsequent analyses, Thy1.2 expression was measured using a different anti-Thy1.2 antibody (clone 5a-8; Caltag) than the one used for depletion, to avoid complications due to epitope masking. Both Thy1.2-enriched and Thy1.2-depleted fractions were stimulated in vitro for analysis of cytokine production.

In vitro stimulation of thymocytes
Thymocytes, either in bulk or separated based on surface phenotype, were stimulated in vitro (2x10⁵/well) with concanavalin A (Con A) at 5 µg/ml in the presence of antigen-presenting cells (APC) obtained from peritoneal exudate cells (PEC). PEC were obtained from the peritoneal lavage of adult control NFS/NCr mice using standard techniques. PEC were -irradiated, plated at 5x10⁵ cells/well of a flat-bottomed 96-well plate and allowed to adhere to the wells for 2 h at 37°C. Non-adherent cells were extensively washed out of the wells and the remaining adherent cells were used as APC. Thymocytes and APC were co-cultured in 96-well tissue culture plates at 37°C in the presence of 5% CO₂. Supernatants were harvested after 24 h of culture and stored at –70°C for subsequent cytokine testing.

Cytokine assay
IL-4 and IFN- were measured by sandwich ELISA using standard procedures (15). Briefly, polystyrene plates (Maxisorb; Nunc, Rochester, NY) were coated with capture antibody [anti-IL-4 (Endogen, Woburn, MA) and anti-IFN- (R & D Systems, Minneapolis, MN)] at 25°C overnight, and blocked with PBS containing 4% BSA and 0.01% Thimerosal. Next, serially diluted recombinant murine cytokines as standards or culture supernatants samples were added in duplicate and incubated at 37°C for 2 h. The plates were washed and incubated with biotinylated secondary murine detection antibodies (anti-IL-4 and anti-IFN-) at 25°C for 1 h. After washing, wells were incubated with streptavidin–peroxidase polymer in casein buffer and incubated at 25°C for 30 min. After washing, commercially prepared substrate (TMB; Dako) was added and incubated at 25°C for 10–30 min. The reaction was stopped with 2N HCl and the absorbance at 450 nm was read on a microplate reader (Molecular Dynamics, Sunnyvale, CA). Cytokine concentration in each sample was calculated based on standard curves generated with recombinant cytokine controls, using SoftPro software (Molecular Dynamics).

	Results

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Characterization of CD4⁺Thy1^– T cells in the thymus
Lymphocytes recovered from the thymi of NFS/NCr mice from 1 week of age to adulthood were analyzed for cell surface expression of the CD4 and Thy1 molecule. As shown in Fig. 1 (left panels), the majority (85–95%) of CD4⁺ cells co-expressed the Thy1 molecule. The CD4⁺Thy1^– population of thymocytes represented 1–3.7% of the total number of live cells. These CD4⁺Thy1^– cells did not express CD8, and based on forward and side scatter parameters were of the same size and granularity as other recovered lymphocytes. The expression levels of a variety of T cell activation and differentiation markers on CD4⁺Thy1^– and CD4⁺Thy1⁺ thymocytes were compared (Fig. 2, left panels). These markers included the TCR-associated CD3 complex and HSA (CD24) which is expressed at high levels on immature but not mature thymocytes (16,17). CD62L, whose expression increases as thymocytes mature and is down-regulated on peripheral T cells after stimulation (18), was also examined. We analyzed the expression of the activation markers CD25 (the IL-2 receptor chain), CD44 and CD69, which are low on the majority of resting thymocytes and mature T cells, and up-regulate in response to activation signals (19–21). Finally, we examined expression of the maturation marker CD45RB, which is expressed at lower levels on memory versus naive T cells (22). CD4⁺Thy1^– cells were found to express higher levels of CD3, and lower levels of HSA and CD62L than CD4⁺Thy1⁺ cells, consistent with their status as mature thymocytes. The expression of CD25, CD44, CD45RB and CD69 was equally low among CD4⁺Thy1⁺ and CD4⁺Thy1^– cells.

View larger version (43K): [in this window] [in a new window]   Fig. 1. High-dose neonatal infection causes enhanced representation of CD4+Thy1– cells in the thymus. Thymocytes from age-matched control (left column), low-dose (middle column) or high-dose (right column) infected mice were analyzed for their expression of CD4 and Thy1 at 1, 2, 3, 6, 9 and 16 weeks p.i. Data were gated for live lymphocytes (based on exclusion of 7AAD and size) and numbers in the dot-plots indicate the percentage of cells in each quadrant. Low-dose infected mice were not analyzed at week 9.

 

View larger version (51K): [in this window] [in a new window]   Fig. 2. Thymocytes from high-dose infected mice differ in their expression of activation markers. Thymocytes from control (left column), low-dose (middle column) or high-dose (right column) infected mice were analyzed 16 weeks p.i. for their expression of the surface antigens CD3, CD25, HSA, CD44, CD45RB, CD62L and CD69. The histograms show the expression level of each marker among CD4+Thy1+ (dashed lines) and CD4+Thy1– (solid lines) thymocytes using the upper right and upper left quadrants shown in Fig. 1 as gates. The numbers above the markers indicate the percentage of CD4+Thy1+ (left number) and CD4+ Thy1– (right number) cells within the marker. Histograms were gated for lymphocyte-sized, 7AAD– cells and were normalized to allow comparison of the peaks. These data are representative of at least three experiments, and are indistinguishable from results obtained from mice 9 and 12 weeks p.i.

 
To determine if functional differences exist between CD4⁺Thy1⁺ and CD4⁺Thy1^– thymocytes, we analyzed these subsets for their capacity to secrete cytokines. CD4⁺CD8^–Thy1^– thymocytes were isolated from 15to 20-week-old naive mice using a MACS-based depletion protocol and their responses were compared to those of both bulk thymocytes and CD4⁺CD8^–Thy1⁺ thymocytes alone. Although the frequency of CD4⁺CD8^–Thy1^– cells in vivo was much lower than that of CD4⁺CD8^–Thy1⁺ cells, in these in vitro experiments equivalent numbers of cells were stimulated. The subsequent secretion of IFN- and IL-4, the prototypical T_h1 and T_h2 cell cytokines respectively, was determined. When stimulated with Con A in the presence of irradiated, syngeneic PEC, both bulk and Thy1⁺ thymocytes fractions secreted IFN- and IL-4. However, when Thy1^– thymocytes were similarly stimulated, they secreted only IL-4 and no detectable IFN- (Fig. 3A and B, left side).

View larger version (38K): [in this window] [in a new window]   Fig. 3. CD4+Thy1– thymocytes display a Th2-like cytokine profile. Unfractionated or fractionated thymocytes from control (N thymus, N Thy1+, N Thy1–) or mice infected at birth with a high dose of Cas (tested at 15–20 weeks p.i.; Inf. Thymus, Inf. Thy1+, Inf. Thy1–) were cultured with Con A as described in Methods, supernatants harvested at 24 h, and tested for IFN- (A) and IL-4 (B) by ELISA. Data represent mean ± SE of three to five mice in three separate experiments. Background IFN- and IL-4 production by APC was <20 pg/ml.

 
CD4⁺Thy1^– T cells are highly represented in the thymi of mice infected with high-dose Cas virus
Newborn mice infected with a high dose of Cas develop a non-protective T_h2 anti-viral response characterized by the production of IL-4 and fail to generate protective T_h1 associated cytotoxic T lymphocyte responses. These mice exhibit lesions in the central nervous system that lead to hind limb paralysis and eventually death by 16–24 weeks post-infection (p.i.). In contrast, infection of neonatal mice with a low dose of Cas results in the development of a T_h1 anti-viral response, with viral clearance and no virus-associated functional sequelae (11). We investigated whether the IL-4-secreting CD4⁺Thy1^– subset of thymocytes identified in naive mice was present following infection of mice with a high dose of Cas.

Thymocytes recovered from mice inoculated with low-dose or high-dose Cas virus were analyzed for the presence of CD4⁺Thy1^– T cells in the thymus, at different times p.i. As shown in Fig. 1 (middle and right panels), the frequency of CD4⁺Thy1^– cells increased in mice infected with a high dose but not a low dose of Cas virus. This increase was first detected at 6 weeks p.i., and by 16 weeks, nearly half of the CD4-expressing and more than one-fifth of all thymocytes were CD4⁺Thy1^–. There was no similar increase in the frequency of Thy1^– cells among either the double-positive or CD8 single-positive thymic subsets; on the contrary, Thy1^– cells in these subsets never exceeded 2–3% in any of the mice studied. Likewise, there was no increase in the frequency of CD4⁺Thy1^– T cells in mice infected with a low dose of virus at any of the time points examined. As found in naive mice (see above), the CD4⁺Thy1^– cells from high-dose infected animals were of the same size and granularity as other recovered thymocytes.

Activation/differentiation markers expressed by CD4⁺Thy1^– T cells from the thymi of high-dose Cas-infected mice
The surface phenotype of the CD4⁺Thy1^– cells from high-dose infected animals was compared to that of similar cells from low-dose infected and naive control mice, as shown in Fig. 2 (right panel relative to middle and left). All of the CD4⁺Thy1^– cells analyzed, regardless of their origin, expressed high levels of CD3, and low levels of CD25, CD45RB and CD69, closely resembling the expression patterns of CD4⁺Thy1⁺ cells. CD4⁺Thy1^– thymocytes from all backgrounds expressed lower levels of HSA and CD62L than CD4⁺Thy1⁺ cells, although these differences were more pronounced among thymocytes from high-dose infected mice. The expression of CD44 was low among CD4⁺Thy1⁺ cells from low-dose and high-dose infected mice, resembling the CD44 expression on CD4⁺Thy1^– cells from control and low-dose infected mice. In contrast, CD4⁺Thy1^– cells from high-dose infected mice uniformly expressed CD44 at levels 3to 4-fold higher than was detected on any other thymocyte population. The phenotype of CD4⁺Thy1⁺ cells from high-dose infected mice resembled that of cells from control mice, with the exception that a higher percentage of these Thy1⁺ cells expressed low levels of HSA (47.2% HSA^lo from high-dose infected versus 4.4% HSA^lo from control mice).

To examine the extent of Cas infection among thymocytes, cells recovered from mice inoculated with low-dose or high-dose Cas virus were analyzed for their cell surface expression of the Cas envelope glycoprotein gp70. As shown in Fig. 4(a), thymocytes from mice infected with a low dose of Cas did not express gp70 at any of the time points examined (as compared to age-matched, uninfected controls). Among thymocytes from high-dose infected mice, significant gp70 surface expression was first detected on a fraction of thymocytes 2 weeks after infection. By 6 weeks p.i., >65% of the recovered thymocytes expressed gp70, suggesting a slowly paced, ongoing infection in the thymus. Interestingly, the distribution of gp70 on the surface of thymocytes was non-Gaussian, appearing as either a very broad peak or two overlapping peaks of gp70 staining, indicating substantial heterogeneity in gp70 surface levels among thymocytes from high-dose infected mice. Thymocytes expressing high levels of CD3 had greater gp70 expression, while the majority of CD3^lo cells had lower levels of gp70 (see Fig. 4b). When co-stained for Thy1, CD4 and gp70 expression, all Thy1^–CD4⁺ thymocytes from high-dose infected mice were found to express high levels of gp70 at all time points after 2 weeks (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Thymocytes from high-dose but not low-dose infected mice express the Cas virus glycoprotein gp70. (A) Expression of gp70 by thymocytes from age-matched control (dotted line), low-dose (thin solid line) and high-dose (thick solid line) infected mice is shown at 1, 2, 3, 6, 9 and 16 weeks p.i. (B) gp70hi cells express high levels of CD3. Using the histogram markers shown in the 16 week panel of (A) to differentiate gp70hi from gp70lo cells, expression of CD3 on gp70hi (thick solid line) and gp70lo (dotted line) thymocytes from high-dose infected mice is shown.

 
High-dose Cas infection leads to accelerated thymic atrophy
Thymus cellularity in mice infected with a high dose of virus was reduced as compared to control mice; this reduction was evident as early as 6 weeks after infection (Fig. 5). After 16 weeks, thymi from mice infected with a high dose of virus had roughly a quarter the number of cells as found in uninfected thymi, and contained a higher percentage of non-lymphoid cells based on flow cytometric forward and side scatter parameters. In some experiments, mice infected 16 weeks earlier were administered an i.p. injection of a dilute solution of India ink 30 min prior to sacrifice. This procedure stains the lymph nodes, but not the thymus, and facilitates the precise isolation of thymic cells by labeling adjacent parathymic lymph nodes. Results from India ink-treated mice were indistinguishable from those obtained from uninjected mice, indicating that thymic samples, even those from atrophied thymi, were not contaminated with lymph node cells.

View larger version (34K): [in this window] [in a new window]   Fig. 5. High-dose Cas infection leads to accelerated thymic atrophy. Thymi were taken from control mice or mice infected with high-dose Cas at 1, 2, 3, 6, 9 and 16 weeks p.i., and single-cell suspensions were prepared from each organ. Cell counts of live thymocytes (based on exclusion of Trypan blue) are presented. Data represent mean ± SE of cell numbers/thymus of two to eight mice for each time point.

 
The CD4 and CD8 expression on thymocytes was examined to determine whether the reduction in thymic cell number reflected a global cell loss or the selective loss of certain thymic subsets. During the first 3 weeks p.i., the majority of cells from mice infected with a high dose of virus did not display significant differences from low-dose or uninfected mice in their expression of CD4 and/or CD8 (Fig. 6). However, between 3 and 6 weeks after infection, the CD4/CD8 profile of thymocytes from high-dose infected mice changed dramatically. Concomitant with the rapid decline in thymus cellularity (see Fig. 5) was a relative increase of up to 9-fold in the frequency of CD4 and CD8 single-positive cells, coupled with a relative decrease of 3to 4-fold in the frequency of CD4/CD8 double-positive cells. Likewise, the relative frequency of double-negative cells was enhanced ~3-fold in thymocytes from high-dose infected mice compared to low-dose infected or naive controls. Together, these results indicate that high-dose Cas infection leads not only to a decrease in the absolute number of cells in the thymus, but also to a selective loss of CD4/CD8 double-positive thymocytes. Nonetheless, the absolute number of CD4⁺Thy1^– cells remained unaltered in the thymi of high-dose infected mice relative to naive mice (compare averages of 2.8% of 8.5x10⁷ cells, or 2.4x10⁶ CD4⁺Thy1^– cells from 16-week-old naive mice to 4.4x10⁶ such cells from atrophied, high-dose infected week 16 thymi).

View larger version (53K): [in this window] [in a new window]   Fig. 6. High-dose neonatal infection alters the distribution of CD4+ and CD8+ thymocytes. Thymocytes as described in Fig. 1 were analyzed for their CD4 and CD8 expression. Each dot-plot is representative of two to four mice, and shows data gated for lymphocyte size and exclusion of 7AAD. Numbers indicate the percentage of cells in each quadrant. Low-dose infected mice were not analyzed at week 9.

 
CD4⁺Thy1^– thymocytes from mice infected with high-dose Cas display T_h2-like cytokine secretion
To determine if high-dose Cas infection influences the capacity of thymocytes to secrete cytokines, IFN- and IL-4 production by thymocytes from high-dose infected mice was examined as described above. Although the frequency of CD4⁺Thy1^– cells in vivo was much lower in control mice than in mice infected with a high dose of virus, in these in vitro experiments equivalent numbers of CD4⁺Thy1^– cells from control or mice infected with a high dose of virus were stimulated to assess cytokine secretion. When treated with Con A, both bulk and Thy1⁺ thymocytes fractions from mice infected with a high dose of virus secreted IFN- and IL-4 (Fig. 3a and b, right side). This indicated that, despite infection with Cas virus and disruption of normal thymocyte development, thymocytes from infected mice remained capable of responding to a mitogenic stimulus by producing both T_h1and T_h2-like cytokines. However, when CD4⁺Thy1^– thymocytes from mice infected with a high dose of virus were stimulated with Con A, they secreted only IL-4 and no detectable IFN-.

	Discussion

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In this study we identified and characterized a small population of CD4⁺CD8^–Thy1^– T cells present in the thymus of naive newborn and adult mice. This Thy1^– subset represents 1–3.7% of the total thymocyte population. CD4⁺Thy1^– thymocytes from naive mice express high levels of CD3, and low levels of CD25, CD45RB, CD69, HSA, CD44 and CD62L. When stimulated in vitro, CD4⁺Thy1^– thymocytes secreted only IL-4 and no detectable IFN-. Finally, thymocytes of this functional and surface phenotype were found to be increased in frequency 10-fold in the thymi of mice infected at birth with a high dose of Cas virus, despite the dramatic thymic atrophy that is associated with high-dose infection. In contrast, mice infected at birth with a low dose of Cas virus displayed neither increased frequency of CD4⁺Thy1^– thymocytes nor thymic atrophy.

In a previous study, we demonstrated that the fate of mice infected as neonates with the Cas virus depended on the type of T cell response elicited (T_h1 or T_h2). This differentiation process was influenced by the initial dose of Cas administered (11). We have now compared the thymi of mice infected with either a high or low dose of Cas virus to determine how Cas infection alters lymphocyte populations and found that after high-dose infection thymocytes express gp70 fairly slowly, such that it was not until 6 weeks p.i. that the majority of cells became positive for gp70. This may reflect a differential ability of the Cas virus to infect thymocytes at different stages of development, as suggested by the finding that thymocytes with high levels of CD3 express gp70 earliest (by week 2 p.i.) and at highest levels (at all time points). Alternatively, infection of thymocytes could be initiated by the re-entry into the thymus of mature T cells that became infected in the periphery. The finding that at 16 weeks after infection roughly half of the CD4 single-positive cells and virtually all of the CD4⁺Thy1^– thymocytes express very low levels of HSA, a phenotype consistent with mature peripheral T cells, supports this possibility. In this regard, it has been shown that in neonates but not adults, a subset of peripheral T cells that express low levels of CD62L can re-enter the thymus (23,24). Significantly, nearly all of the CD4⁺Thy1^– cells in the thymus at 16 weeks p.i. express high levels of gp70 and low levels of CD62L.

It remains unclear how high-dose Cas infection causes accelerated thymic atrophy and what role (if any) this thymic atrophy plays in disease progression. Peripheral T cell numbers do not drop as a consequence of the (presumably) decreased thymic output of newly arisen T cells; on the contrary, splenic and lymph node populations of T cells actually expand 2- to 3-fold by 16 weeks after high-dose Cas infection (data not shown). It may be that thymic atrophy after high-dose Cas infection reflects either direct toxicity or enhanced emigration of thymocytes, with double-positive cells, which are selectively depleted, being most susceptible to the effect. As a non-mutually exclusive possibility, infection of thymic stromal cells could result in either alterations or ablation of their normal support functions, inhibiting thymocyte expansion and maturation.

Likewise, the reason for the selective expansion/persistence of CD4⁺Thy1^– T cells in the thymus after high-dose Cas infection is unresolved. Thy1 is a glycosylphosphatidylinositol-linked membrane protein and as such is down-regulated on the surface of most activated T cells through the activity of the phosphatidylinositol-specific enzyme phospholipase C (25,26). It is possible that the lack of Thy1 expression on the thymocytes described here reflects a state of chronic activation, perhaps in response to Cas viral antigens. However, CD4⁺Thy1^– T cells do not express low levels of the CD3–TCR complex, as would be expected of cells chronically stimulated by peptide–MHC complexes. Furthermore, the CD4⁺Thy1^– cells from high-dose infected mice do not express appreciable levels of either CD25, the high-affinity IL-2 receptor, or CD69, an early lymphocyte activation marker. They do, however, express high levels of CD44 and low levels of CD62L; this phenotype is usually associated with memory T cells (27). Together, these results suggest that the lack of Thy1 expression on these CD4⁺ T cells reflects an altered differentiation state rather than a response to stimulation. In this regard, it is important to note that CD4⁺Thy1^– T cells are also present (at reduced frequency) in the thymi of age-matched, uninfected control mice and thus represent a naturally occurring subset of T cells. It may be that this subset of cells is uniquely resistant to thymic atrophy, as has been suggested in other experimental systems (6).

Other groups have reported the presence CD4⁺Thy1^– T cells. As mentioned above, infection of mice with a different murine retrovirus (LP-BM5) has been shown to result in the selective expansion of peripheral CD4⁺Thy1^– T cells (5). The role of these CD4⁺Thy1^– T cells in the progression of MAIDS has not been established, although phenotypically these peripheral Thy1^– cells resemble the thymic cells described here in that both populations express very high levels of CD44. CD4⁺Thy1^– T cells are also over-represented in mice either recovering from sub-lethal irradiation or from irradiation followed by bone marrow transplantation (6,7). In these cases, it is unclear whether the CD4⁺Thy1^– T cells are of thymic or extrathymic origin, and the cytokine secretion capacity of these cells was not examined. The T cells localized in gut associated Peyer's patches are greatly enriched for CD4⁺Thy1^– T cells (8), as are T cells in the germinal centers of spleen and lymph nodes (9). Interestingly, germinal center T cells have been found to express high levels of IL-2 and IL-4 but not IFN- mRNA in situ. (28). In the current study it is unlikely that the emergence of CD4⁺Thy1^– thymocytes reflects aberrant germinal center formation in the thymus, since the frequency of B220⁺ B cells never exceeded 2% in any of the thymi analyzed and the few B cells present did not express the germinal center marker GL-7. A CD4⁺Thy1^– T cell line established from Peyer's patch T cells secreted IL-2, small amounts of IL-4 and IL-5, but not IFN-, in response to stimulation with an anti-CD3 antibody (8). Together, these studies suggest a generalized T_h2-like cytokine profile for CD4⁺Thy1^– T cells.

The persistence of CD4⁺Thy1^– T cells accompanied by the decrease in total cell number in the thymi of mice infected with a high dose of virus may indicate a direct role for these cells in the Cas virus-induced disease process. The secretion of IL-4 but not IFN- by CD4⁺Thy1^– T cells in response to Con A indicates that these cells have either differentiated or are in some way intrinsically programmed to be T_h2-like cells. It is therefore interesting and potentially significant that neonatal infection with high-dose Cas virus leads to a virus-specific, non-protective T_h2-type T cell response. The CD4⁺Thy1^– cells (or at least a significant subset thereof) that are found in the thymi of high-dose infected mice may in fact be specific for Cas viral antigens. Alternatively, these cells may play a role in driving virus-specific T cells into a T_h2 phenotype after high-dose Cas infection and may help unraveling the catalytic role of IL-4-producing cells (reviewed in 29) in the induction of polarized T_h2 responses in vivo. We are currently investigating the antigen specificity of these CD4⁺Thy1^– cells, their capacity to arise in other strains of mice and their contribution to Cas-mediated disease.

	Acknowledgments

 
We thank Shui Cao for his excellent assistance and the Cytokine Core Facility of the University of Maryland, Baltimore, MD for performing the cytokine ELISA. This work was supported by NIH Grants CA65388 (to M. S.), AI24335 and AG13789 (to G. K.)

	Abbreviations

 

7AAD 7-aminoactinomyosin D

APC antigen-presenting cell

Cas Cas-Br-E MuLV

Con A concanavalin A

HSA heat-stable antigen

MuLV murine leukemia virus

PEC peritoneal exudate cell

p.i. post-infection

	Notes

 
Transmitting editor.Z. Ovary

Received 26 June 2000, accepted 4 October 2000.

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