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The Journal of Immunology, 2000, 165: 1896-1901.
Copyright © 00 by The American Association of Immunologists

{alpha}ßTCR+ Cells Are a Minimal Fraction of Peripheral CD8+ Pool in MHC Class I-Deficient Mice1

Dragana Nesic2, Fabio R. Santori and Stanislav Vukmanovic3

Michael Heidelberger Division of Immunology, Department of Pathology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of ß2-microglobulin-deficient (ß2m-/-) mice are unknown. We here show that very few CD8+ cells in ß2m-/- mice coexpress CD8ß, a marker of the thymus-derived CD8+ T cells. Most of the CD8{alpha}+ cells express CD11c and can be found in ß2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8{alpha}+CD8ß+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8ß+ cells in ß2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in ß2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8{alpha} become prominent when CD8+ T cells are virtually absent; 3) at least some ß2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class I molecules play a crucial role in CD8+ T lymphocyte differentiation. Numerous approaches have confirmed the role of MHC class I in positive selection in the thymus. These include early bone marrow chimera experiments (1, 2, 3), the use of anti-MHC class I Abs to block the development of CD8+ T cells (4), the use of MHC class I-restricted TCR-transgenic mice (5, 6), and development of MHC class I-deficient mice models showing impaired positive selection of CD8+ T cells (7, 8). In addition to the well-established role in positive selection in the thymus, it has recently been recognized that MHC class I molecules also play a significant role in maintaining naive CD8+ T cells in the periphery, as few, if any, CD8+ cells survived after adoptive transfer into the MHC class I-deficient hosts (9) or after emigration from the MHC class I-expressing thymus into the MHC class I-deficient periphery (10). CD8+ cells in the absence of peripheral MHC class I first down-regulate CD8 expression becoming CD4-CD8- and then undergo apoptosis mediated by the Fas-Fas ligand interaction (11).

Despite impairment of both thymic and peripheral selection, residual CD8+ T cells are present in ß2-microglobulin (ß2m)4-deficient (ß2m-/-) mice as evidenced by rejection of ß2m+/+ allogeneic tumors in a CD8-dependent manner (12, 13) and detection by flow cytometry or by the CD8-dependent cytotoxicity to MHC class I-positive targets of expanded TCRß+CD8{alpha}+ cells upon immunization with ß2m+/+ tumor or spleen cells (12, 14, 15). CD8{alpha}+ cells are present in ß2m-/- mice even before tumor cell injection, and their frequency is about 50-fold lower in the peripheral blood (16) or 10- to 20-fold lower in the spleens (10) compared with wild-type mice. In total, the numbers of CD8+ cells are estimated to be ~2 x 106 per ß2m-/- mouse (11), compared with ~40 x 106 normally found in wild-type mice (17).

There are at least two aspects of the ß2m-/- CD8+ compartment that make it qualitatively distinct, rather than a ~20-fold reduced copy of the wild type. First, alloreactive CD8+ responses cannot be raised from ß2m-/- lymphoid tissues without prior in vivo immunization (12, 14, 15). Assuming a similar frequency of alloreactive TCRs within ß2m-/- and wild-type CD8+ cells (the frequency of alloreactive cells is on average 1–10% in wild-type mice (18)), the frequency of alloreactive CD8+ cells in total ß2m-/- spleen should be 5–100/104 cells. Even 10-fold lower frequencies (5–100/105 spleen cells) of Ag-specific CD8+ cells from primed ß2m+/+ mice readily produce detectable cytolytic responses after in vitro restimulation with Ag (19). Thus, allospecificity is aparently absent from the ß2m-/- CD8+ repertoire, and this could be explained by three possibilities: 1) the TCR{alpha}ß repertoire of CD8+ cells in ß2m-/- mice may be biased against allorecognition; 2) ß2m-/- CD8+ T cells are functionally nonresponsive; or 3) the majority of CD8+ cells in ß2m-/- mice may not belong to the TCR{alpha}ß+ T cell lineage, but some other T or non-T cell lineage.

The second characteristic of peripheral ß2m-/- CD8+ cells is that they undergo high frequency of apoptosis (2- to 3-fold over the levels in wild-type CD8+ cells) when cultured in vitro, presumably due to the absence of survival signals provided by peripheral MHC class I (11). To maintain the apoptosis-prone CD8+ compartment, the input of fresh CD8+ cells must be relatively increased. However, the thymus in ß2m-/- mice is literally devoid of CD8+ T cells, both phenotypically (7, 8) and functionally (10). Thus, there is a discrepancy between the apparently required increased thymic export of CD8+ T cells and their virtual absence in the thymus. Two possible explanations could account for this apparent paradox: 1) there is a compensatory increase in the output of ß2m-/- CD8+ cells from the thymus with a rapid transition of mature CD8+ T cells through the thymic medulla (20); and 2) the majority of CD8+ cells in noninjected ß2m-/- mice may not belong to the TCR{alpha}ß+ T cell lineage, but some other T or non-T cell lineage. Thus, the two curios features of the ß2m-/- CD8+ cell compartment could both be explained by different origin of ß2m-/- CD8+ cells. Indeed, we here provide evidence that most of the ß2m-/- CD8+ cells are not mainstream {alpha}ßTCR+ cells. We also demonstrate that the mainstream ß2m-/- CD8+ T cells have a reasonably long life span and do not require wild-type MHC class I in the periphery for the survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, cells, and reagents

Five- to 7-wk-old female ß2m-/- mice (C57BL/6 background), recombination-activating gene (RAG)-2-/- (C57BL/6 and BALB/c backgrounds), and wild-type C57BL/6 mice were purchased from Taconic (Germantown, NY). TCRß-deficient mice (21) were a kind gift from Dr. Juan Lafaille (New York University School of Medicine). ß2m/RAG-2 double-deficient mice were obtained as an F2 generation between ß2m-/- (C57BL/6 background) and RAG-2-/- (C57BL/6 x BALB/c)F1 and were screened by peripheral blood immunofluorescent staining for the presence of CD4+ cells and MHC class I. Generation and maintenance of long-term P815-specific CD8+ cell lines were previously described (13, 15). The following mAbs were used: PE- or CyChrome-conjugated anti-CD8{alpha} (53-6.7; PharMingen, Costa Mesa, CA), FITC-conjugated anti-CD8{alpha} (3.168; prepared in our laboratory), FITC-conjugated anti-CD8ß-chain (53-5.8; PharMingen), PE-conjugated anti-CD4 (H129.19; PharMingen); FITC-conjugated anti-TCRß-chain (H57-597, prepared in our laboratory); FITC-conjugated anti-H-2Kd (SF1-1.1; PharMingen) biotinylated anti-TCR{delta} (GL3; PharMingen); PE-conjugated anti-CD11b (M1/70; PharMingen); PE-conjugated anti-CD11c (HL3; PharMingen); PE-conjugated anti-NK1.1 (PK136; PharMingen); and unconjugated anti-H-2Kb (prepared in our laboratory).

Adult thymectomy, generation of bone marrow chimeras, and thymus grafting

Adult thymectomy was performed aseptically under general avertin anesthesia. Sternum was exposed and cut in its upper third, and the thymus was removed using a suction device. Mice were allowed to recover for 2 wk and were then injected s.c. with 1 x 106 live P815 cells. Four weeks later, the mice were boosted with an i.p. injection of 1 x 107 irradiated P815 cells. Analysis was performed 5 days after the second injection. At that point, the success of thymectomy was verified by inspection, and only mice with completely removed thymus were included in the study. To generate bone marrow chimeras, ß2m-/- mice were lethally irradiated (800 rad) and immediately reconstituted by i.v. injection of 5 x 106 ß2m+/+ or ß2m-/- bone marrow cells. Mice were left to recover for 4 wk and then grafted with 5-deoxyguanosine-treated ß2m+/+ neonatal thymi as previously described (10).

Flow cytometry

Spleen or lymph node cells were single, double, or triple stained using PE- or FITC-conjugated anti-CD8{alpha} mAb and a second FITC-, PE-, or biotin-conjugated Ab of various specificities. Cells were incubated 20 min at room temperature with 20% normal mouse serum in PBS (1% BSA). mAbs were then added and cells were incubated on ice for 30 min and washed in PBS (1% BSA). If biotin-conjugated Ab was used, cells were incubated on ice another 30 min with a 1/100 dilution of CyChrome-streptavidin (PharMingen). Cells were then fixed in 1% paraformaldehyde and analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). For coexpression studies, cells positive for CD8{alpha} were electronically gated at the acquisition level, and the expression of another molecule on gated cells was analyzed using CellQuest software (Becton Dickinson). Control cells were stained with anti-CD8{alpha} Ab alone (and CyChrome-streptavidin, where appropriate) and gated of CD8{alpha}+ cells in an identical manner. Single-color staining for H-2Kb was performed as previously described (22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Majority of CD8{alpha}+ cells in ß2m-/- mice do not express CD8ß

There is a distinct lineage of TCR{alpha}ß T cells that are thought to mature in the intestine, many of which are of CD8+CD4- phenotype (23, 24). In addition, cells of other lineages can express the CD8 molecule, such as NK cells (25), TCR{gamma}{delta} T cells (26), or dendritic cells (27). NK cells are functionally deficient, but physically present in ß2m-/- mice (28). Any of these cell types, or a combination of them, could potentially represent significant portion of the CD8+ cell pool in ß2m-/- mice. A common denominator of all these cells is the form of the CD8 molecule they express: all of them express a CD8{alpha} homodimer (23, 24), while thymus-derived TCR{alpha}ß cells express a heterodimer consisting of CD8{alpha}- and CD8ß-chains. Thus CD8ß expression could be used to infer the origin of the CD8+ cells. Spleen cells from ß2m+/+ or ß2m-/- mice were stained with PE-conjugated anti-CD8{alpha} and FITC-conjugated anti-CD8ß. The acquisition gate was set to include only anti-CD8{alpha}-labeled cells, and the expression of CD8ß on gated cells was analyzed. In contrast to the wild-type mice, only ~8–9% of CD8{alpha}+ cells coexpressed CD8ß in the spleens of ß2m-/- mice (Fig. 1Go), suggesting that the majority of CD8{alpha}+ cells in ß2m-/- spleens are not thymus derived. In addition to the CD8ß expression, ß2m+/+ and ß2m-/- cells differed with respect to their size. A significant proportion of CD8{alpha} cells in ß2m-/- mice consisted of relatively large cells (Fig. 1Go), hence the more intense background staining in ß2m-/- cells.



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  		FIGURE 1. Distinct phenotype of ß2m-/- or TCRß-/- splenic CD8{alpha}+ cells. Wild-type, ß2m-/-, or TCRß-/- spleen cells were stained with PE-conjugated anti-CD8{alpha} and FITC-conjugated anti-CD8ß mAbs and analyzed by flow cytometry. The acquisition gate was set to include only CD8{alpha}-positive cells (PE), and these were then further analyzed for cell size and the expression pattern of CD8ß. The numbers indicate the percentage of positive cells defined by the indicated marker. For the CD8ß expression, the number was obtained after the percent of control cells (plain line) in the marker area has been subtracted from the percent cells stained with the mAb (bold line).

 
It could be argued that the CD8{alpha}+ cells in ß2m-/- mice are in fact mainstream {alpha}ßTCR+ cells but appear CD8ß- due to a potentially faster clearance of CD8ß-chain compared with the CD8{alpha}-chain, both caused by the absence of MHC class I (11). TCR{alpha}-/- and/or TCRß-/- mice should address this issue as they express MHC class I, but do not allow any thymic selection of T cells in the thymus. Presence of cells of a similar phenotype (CD8{alpha}+CD8ß-) in TCR{alpha}-/- and TCRß-/- mice would exclude the above-mentioned possibility. Indeed, the majority of CD8{alpha}+ cells in both TCR{alpha}-/- (not shown) and TCRß-/- spleens are CD8ß- (Fig. 1Go). Thus, the absence of CD8ß expression by most CD8{alpha}+ cells in ß2m-/- mice could be explained better by an {alpha}ßT cell deficiency, or perhaps more precisely CD8+ T cell deficiency rather than MHC class I deficiency.

Lineage marker expression by ß2m-/- CD8{alpha}+ cells

To determine the lineage(s) of the majority of ß2m-/- CD8+ cells, further immunofluorescent analyses of ß2m-/- spleen cells were performed. These studies indicated that virtually none of the ß2m-/- CD8{alpha}+-gated cells expressed the NK1.1 marker, although there was a distinct population of NK1.1+ cells in total ß2m-/- spleen cell population (Fig. 2Go). Approximately 20% of CD8{alpha}+ cells expressed each TCRß and TCR{delta}, leaving the origin of the majority of CD8{alpha}+ cells still unaccounted for.



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  		FIGURE 2. Lineage marker expression by ß2m-/- CD8{alpha}+ cells. ß2m-/- spleen cells were stained with FITC- or PE- conjugated anti-CD8{alpha} and a second Ab marker specific for NK cells, T cells, or dendritic cells. Abs used were PE-conjugated anti-NK1.1, biotinylated anti-TCR{delta} (followed by streptavidin-CyChrome), FITC-anti-TCRß, or PE-anti-CD11b. The acquisition gate was set to include only CD8{alpha}-positive cells, except in lower left panel where total cells are analyzed as a reference for positive staining with anti-NK1.1 Ab. Gated CD8{alpha}+ cells were further analyzed for the expression of the other markers. The numbers indicate the percentage of positive cells defined by the marker shown. This number was obtained after the percent of control cells stained only with anti-CD8{alpha} Ab (plain line) in the marker area has been subtracted from the percent cells stained with a given mAb (bold line).

 
To determine whether any CD8{alpha}+ cells in ß2m-/- mice might belong to the dendritic cell lineage, we first determined the expression of CD11b, a marker of the macrophage/dendritic cell lineage (29, 30). Strikingly, more than half of the cells stained positive (Fig. 2Go). To test the dendritic origin of these cells more rigorously, we examined the expression of CD11c, which is a more specific marker of dendritic cells (30). An almost identical percentage (~55%) of CD8{alpha} cells stained with CD11c (Fig. 3GoA). A three-color immunofluorescent analysis indicated that the expression of CD11c and CD8ß was mutually exclusive (Fig. 3GoA), and that CD11c+ cells, and not CD8ß+ cells, contained relatively large cells (Fig. 3GoB). When ß2m-/- mice were bred to RAG-2-/- mice to eliminate all DNA rearrangement-dependent cells, CD8ß+CD11c- were depleted while CD8ß-CD11c+ were relatively enriched in double-deficient offsprings (Fig. 3GoC). Cellularity of ß2m-/-/RAG-2-/- spleens was ~10-fold lower than that of ß2m-/-/RAG-2+/- mice (data not shown), so the absolute number of CD11c+CD8{alpha}+CD8ß- cells was in effect also reduced in double-deficient mice. Also, of interest to note are the relatively higher levels of CD8{alpha}+CD8ß+ cells in the ß2m-/-/RAG-2+/- mouse in this experiment. This was most likely due to the mixed BALB/c x C57BL/6 background imposed by the nature of breeding we performed (see Materials and Methods). Taken together, it can be concluded that most (50–60%) of the splenic CD8+ cells in ß2m-/- mice are most likely dendritic cells.



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  		FIGURE 3. The majority of CD8{alpha}+CD8ß- cells are dendritic cells. A, ß2m-/- spleen cells were triple stained with CyChrome-conjugated anti-CD8{alpha}, FITC-conjugated anti-CD8ß, or FITC-conjugated anti-H-2Kd (negative control) and PE-conjugated anti-CD11c or PE-conjugated anti-NK1.1 (negative control). The acquisition gate was set to include only CD8{alpha}-positive cells, and the expression of the other two molecules is shown. The numbers indicate the percentage of positive cells defined by the markers shown. B, Cell size of CD8{alpha}+CD11c+CD8ß- and CD8{alpha}+CD11c-CD8ß+ cells. C, The same analysis as in A performed on spleen cells from ß2m-/- (upper panel) or ß2m-/-/RAG-2-/- mice (lower panel).

 
The abundance of CD8{alpha}+ dendritic cells could be somehow induced in ß2m-/- mice, or alternatively the paucity of normally dominant mainstream CD8{alpha}+CD8ß+ T cells could have merely exposed CD8{alpha}+ dendritic cells to a prominent level. If the latter is the case, then a CD8{alpha}+CD8ß-CD11c+ population should exist in wild-type mice, too. Indeed, a portion (0.6%) of wild-type spleen cells are CD8{alpha}+CD8ß-, and when gates were set to enrich for this population a third color analysis revealed that almost half of these cells expressed CD11c (Fig. 4Go). Thus, although mildly more frequent in ß2m-/- mice, CD11c+ cells are a major component of CD8{alpha}+CD8ß- population in wild-type mice, too.



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  		FIGURE 4. Significant portion of CD8{alpha}+CD8ß- cells in wild-type mice are dendritic cells. Wild-type C57BL/6 spleen cells were triple stained with CyChrome-conjugated anti-CD8{alpha}, FITC-conjugated anti-CD8ß, and PE-conjugated anti-CD11c or PE-conjugated anti-NK1.1. The percentage of CD8{alpha}+CD8ß- cells in the total population is shown in the upper left plot. These cells were subsequently gated at the acquisition level (lower left plot), and the expression of NK1.1 (upper right plot) or CD11c (lower right plot) vs cell size is shown. The number indicates the percentage of cells defined by the gate.

 
CD8ß+ ß2m-/- T cells selectively expand in response to tumor challenge

Immunization of ß2m-/- mice with ß2m+/+ cells results in increased accumulation of TCRß+CD8{alpha}+ cells (14, 15, 31) and induction of MHC class I-directed CTL activity (12, 14, 15, 16, 31, 32, 33). In addition, CD8{alpha}+ cell lines can be generated by repeated in vitro restimulations with ß2m+/+ tumors (15). Given the virtual absence of CD8ß+ T cells in the spleens of naive ß2m-/- mice, we wondered whether functional response and enrichment of the CD8+ compartment after immunization with tumor was due to expansion of thymus-derived CD8+ T cells or whether extrathymic CD8+ cells may account for tumor injection-induced changes. The high levels of CD8ß expression by P815-specific cell lines (Fig. 5Go) argue that tumor injection stimulated CD8+ T cells of thymic origin. Thus, although barely detectable by FACS staining in unmanipulated ß2m-/- spleens, immunization with ß2m+/+ tumors can induce sufficient accumulation of thymus-derived CD8+ cells.



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  		FIGURE 5. CD8ß is expressed by long-term alloreactive ß2m-/- CD8+ cells. Long-term P815-specific TCR{alpha}ß+CD8{alpha}+ cell line (data not shown) stained with anti-CD8ß-FITC mAb (bold line) or left unstained (plain line) was analyzed by flow cytometry.

 
TCRß+CD8ß+ reside in the periphery of ß2m-/- mice for at least 2 wk

ß2m-/- CD8+ cells die in vitro at a rate of ~70%/48 h compared with the ~20% rate of ß2m+/+ CD8+ cells (11). However, the rates of apoptosis may be due to the different cellular origin of CD8+ cells between the wild-type and ß2m-/- mice. Thus, the question of the life span of thymus-derived CD8+ cells in ß2m-/- mice remains open. Lymphocyte life span is normally determined by in vivo 5-bromo-2'-deoxyuridine labeling followed by double staining using lymphocyte subset-specific and 5-bromo-2'-deoxyuridine-specific Abs (17). However, the minimum fraction of CD8ß+ cells in ß2m-/- spleens as detected by immunofluorescence (Figs. 1Go and 3Go), makes this approach impossible to apply to the ß2m-/- mouse model. Instead, we used tumor injection-induced rescue of CD8ß+ cells as a measure of their peripheral survival. We thymectomized adult ß2m-/- mice to remove the supply of new thymic emigrants and injected the mice with P815 cells 2 wk after thymectomy. With a potentially high rate of apoptosis, CD8ß+ cells should be virtually nonexistant by any means of detection by the end of a 2-wk period. However, there was a minor increase in the numbers of CD8ß+ cells ex vivo, and this increase, although small, was significant because after an additional in vitro restimulation with Ag, CD8ß+ cells were abundant (Fig. 6Go). This experiment suggests that a small fraction of CD8ß cells resided for at least 2 wk (time lapsed between the thymectomy and immunization) in the spleens of ß2m-/- mice.



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  		FIGURE 6. CD8ß+ cells can be detected in spleens of thymectomized ß2m-/- mice following stimulation with alloantigen. ß2m-/- mice were thymectomized and injected with P815 cells 2 wk later. Spleen cells were restimulated in vitro with P815 cells and then stained for CD4 vs CD8ß (left). For a comparison, ß2m-/- mice were treated equally except that in vivo immunization was omitted (right).

 
Wild-type MHC class I expression by both thymic epithelium and bone marrow-derived cells is required for the peripheral CD8+ T cell compartment

Very small numbers of CD8+ cells injected into immunodeficient hosts proliferate and can significantly reconstitute the peripheral CD8+ T cell compartment (34). This proliferation is dependent on the presence of MHC class I molecules in the periphery (35). We wanted to determine whether small numbers of CD8+ cells selected by the ß2m-/- thymus could expand in the periphery if provided with the wild-type MHC class I-expressing bone marrow-derived cells. ß2m-/- mice were irradiated and were reconstituted with ß2m+/+ bone marrow. As a control, a second group of irradiated ß2m-/- mice received ß2m-/- bone marrow. The presence of class I-expressing cells in the peripheral blood was demonstrated by staining with anti-H-2Kb Ab (Fig. 7GoA). Each group of bone marrow recipients was either grafted 4 wk later with ß2m+/+ thymic epithelium to allow generation of mature CD8+ T cells or left ungrafted. Two months after the thymus grafting, we analyzed the manipulated animals for the presence of CD8+ cells. Only mice that were transplanted with both ß2m+/+ bone marrow and thymus showed a significant increase in the peripheral CD8+ cell compartment. That was evident in both spleen (data not shown) and lymph nodes (Fig. 7GoB). In all other experimental groups, the periphery was almost devoid of peripheral CD8+ T cells. Thus, over a total period of 12 wk since bone marrow was reconstituted, a <0.5% increase in CD8ß+ T cells could be ascribed to the rescue by the presence of wild-type MHC class I. These findings demonstrate that there needs to be a match between the thymic epithelial and peripheral MHC class I for generation of a sizeable peripheral CD8+ T cell compartment, suggesting that similar ligands induce peripheral homeostasis and positive selection in the thymus.



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  		FIGURE 7. Reconstitution of peripheral CD8+ T cell compartment in ß2m-/- mice by grafting ß2m+/+ bone marrow and thymic epithelium. A, An immunofluorescence analysis of H-2Kb expression by thymocytes of ß2m-/- mice grafted with ß2m+/+ (ß2m+/+BM) or ß2m-/- (ß2m-/-BM) bone marrow and ß2m+/+ thymic epithelium graft (TG). B, CD4 vs CD8ß staining of the lymph node cells of the mice shown in A, The percent of CD8ß+ cells is indicated. Almost identical results were obtained in all three mice per experimental group. Shown are the plots of individual mice from each of the group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show in this report that the CD8{alpha}+ population in ß2m-/- mice is a mixture of various cell types. The common finding for these cells is that most do not express CD8ß. Given that the CD8 molecule can be expressed either as an {alpha}ß heterodimer or {alpha}{alpha} homodimer (36), ß2m-/- CD8{alpha}+ cells most likely express the {alpha}{alpha} homodimer form. Most CD8{alpha}+ cells in ß2m-/- mice express high levels of CD11c and CD11b markers. Neither CD11b nor CD11c are exclusive markers of dendritic cells, and classification of these cells as dendritic cannot be made solely based on the expression of one of them. However, the combination of the high expression of CD11c and the low expression of CD11b and CD8{alpha} is in general diagnostic of dendritic cells (30, 37).

A crucial question is if the presence of these cells is an artifact of the ß2m deficiency or whether they are present in wild-type mice as well. CD8{alpha} expression by mouse spleen dendritic cells has been described (27). We have consistently observed a lower percentage of cells stained with anti-CD8ß Ab than with anti-CD8{alpha} in both the spleens and lymph nodes of C57BL/6 mice (data not shown). In fact, triple staining using anti-CD8{alpha}, anti-CD8ß, and anti-CD11c Abs revealed a distinct population of CD8{alpha}+CD8ß- cells (~8–10% of total CD8{alpha}+ cells or ~0.5% of total spleen cells), and approximately half of these cells expressed CD11c (Fig. 4Go). Thus, CD8{alpha}+CD8ß-CD11c+ cells are not an artifact of ß2m deficiency. In fact, cells with identical phenotype were recently described in bone marrow and were reported to have a facilitating role during bone marrow engraftment (38). Whether the normal role of these cells is to down-modulate or stimulate immune responses is being debated (37).

Recent studies have suggested that peripheral survival of CD8{alpha}+CD8ß+ cells is dependent on constant periodic recognition of self MHC class I (9, 10). Grafting ß2m+/+ thymi into ß2m-/- mice results in positive selection and export of CD8{alpha}+CD8ß+ cells into the periphery of ß2m-/- mice (10). However, such cells are unable to accumulate in the periphery, unless a source of ß2m+/+ cells is provided (Ref. 10 and Fig. 7Go). These findings raise the question of the mechanisms of recruitment, maintenance, and life span of residual peripheral CD8{alpha}+CD8ß+ cells in the ß2m-/- mice. Studies using TCR-transgenic mice suggest that the same restriction element (and therefore possibly the same ligand(s)) induce both thymic-positive selection and peripheral survival (9). In addition, identical ligands that induce positive selection can induce proliferation of CD8{alpha}+CD8ß+ cells transferred into lymphopenic hosts (35). If this is applied to the ß2m-/- mice, then one would speculate that CD8{alpha}+CD8ß+ cells that are selected by the ß2m-free MHC class I in the ß2m-/- thymus should also receive a survival signal from ß2m-free MHC class I molecules in the periphery. Our results showing no additional effect of peripheral expression of wild-type MHC class I on the recruitment of CD8ß+ cells (Fig. 7Go) and the presence of CD8ß+ cells in the periphery 2 wk after thymectomy (Fig. 6Go) are consistent with this view and in disagreement with the suggestion that peripheral ß2m-/- CD8{alpha}+CD8ß+ cells are rapidly dying because of a lack of peripheral MHC class I (11). A steady-state maintenance of peripheral CD8+ T cells with such a high apoptotic rate (11) would require compensatory increased thymic output. However, ß2m-/- thymus is practically devoid of CD8{alpha}+ cells (7, 8). Differences in the apoptotic rates between ß2m-/- and ß2m+/+ CD8{alpha}+ cells should most likely be attributed to the different cellular composition of the CD8{alpha}+ compartment. The fact that true thymus-derived CD8{alpha}+CD8ß+ cells can be found in ß2m-/- mice 2 wk after thymectomy suggests that the life span of ß2m-/- CD8{alpha}+CD8ß+ T cells is likely similar to that of their wild-type counterpart cells.

In conclusion, the major surprise of this study was the finding that mainstream thymus-dependent CD8{alpha}+CD8ß+TCR{alpha}ß+ are barely detectable in ß2m-/- spleens by flow cytometry. They can be induced by antigenic stimulation even 2 wk after adult thymectomy, arguing for their relatively long life span in the periphery. However, they cannot be rescued by syngeneic wild-type MHC class I expressed in the periphery, unless wild-type MHC class I-expressing thymic epithelium is also provided. In the apparent absence of FACS-detectable thymus-derived CD8{alpha}+CD8ß+ cells, the peripheral CD8{alpha}+ compartment comprises of a variety of cell types, most abundant being the ones expressing CD11b/CD11c.


   Acknowledgments
 
We thank Juan Laffaille, Moriya Tsuji, Alan Frey, and Yang Liu for reagents and John Hirst for the flow cytometry analysis.


   Footnotes
 
1 This work was supported in parts by the Markey Charitable Trust Junior Investigator Award and National Institutes of Health Grant AI041573 (to S.V.) and National Cancer Institute Core Support Grant 5P30 CA16087. D.N. was a Jeannette Greenspan Fellow in Cancer Research of the Kaplan Cancer Center. Back

2 Current address: Laboratory of Molecular Immunology, Public Health Research Institute, New York, NY 10016. Back

3 Address correspondence and reprint requests to Dr. Stanislav Vukmanovic, Michael Heidelberger Division of Immunology, Department of Pathology, New York University Medical Center, 550 First Avenue, New York, NY 10016. Back

4 Abbreviations used in this paper: ß2m, ß2-microglobulin; RAG, recombination-activating gene. Back

Received for publication September 21, 1999. Accepted for publication June 5, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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