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The Journal of Immunology, 2001, 166: 5522-5529.
Copyright © 2001 by The American Association of Immunologists

Lymphocyte Lineages at Mucosal Effector Sites: Rat Salivary Glands1

Nancy L. O’Sullivan2,*,{dagger}, Cheryl A. Skandera{dagger} and Paul C. Montgomery{dagger}

Departments of * Anatomy and Cell Biology and {dagger} Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Development of T cell lineages and the role of the thymus as a source of immature T cells in parotid (PG) and submandibular salivary glands (SMG) were studied in Fischer 344 rats using the Thy-1/CD45RC/RT6 expression model. In addition, the phenotypes of salivary gland lymphocytes were compared with other conventional and extrathymic populations. PG mononuclear cells consisted of T cells (38%), B cells (29%), and NK cells (4%). SMG had 19% T cells, 7% B cells, 37% NK cells, and an unusual population of CD3-/RT6+ cells. In comparison with lymph node (LN), both PG and SMG were enriched in immature (Thy-1+) and activated (Thy-1-/CD45RC-/RT6-) T cells. Unchanged percentages of Thy-1+ T cells in PG and SMG following short-term adult thymectomy indicated that immature salivary gland T cells had an extrathymic source. In contrast, thymectomy eliminated LN recent thymic emigrants. SMG had T cells with characteristics of extrathymic populations, expressing TCR{gamma}{delta}+ (28%), the CD8{alpha}{alpha} homodimer (11%), and NKR-P1A (66%). Many SMG T cells expressed integrin {alpha}E{beta}7. PG T cells resembled those isolated from LN in respect to TCR and CD8 isoform usage, but were enriched in {alpha}E{beta}7+ T cells and in NKT cells. Thus, salivary gland mononuclear cells are composed of a variety of subpopulations whose distributions differ between SMG and PG and are distinct from LN. These studies provide a basis for further investigation of regionalization in the mucosal immune network and are relevant to the design of vaccine regimens and intervention during pathological immune processes.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Salivary glands (SG)3 are important effector sites in the mucosal immune network that possess lymphocyte populations, distinct from those in peripheral lymphoid sites, which regulate and mediate humoral and cellular immune responses contributing to the protection of oral surfaces (1, 2, 3, 4). Furthermore, SGs are sites of lymphocytic infiltration in inflammatory disorders such as primary and secondary Sjögren’s syndrome.

Of particular interest is the presence of lymphocytes expressing Thy-1, which, in the rat, is found on immature B and T lymphocytes in bone marrow and thymus (5, 6) as well as on their recently released progeny in the periphery (7, 8). In the case of T cells, the developmental stages of immature recent thymic emigrants (RTE), mature but Ag-naive common peripheral T cells, as well as recently activated T effector cells and memory cells can be identified by their expression of Thy-1, CD45RC, and RT6.

T cell populations of extrathymic origin have been identified and are the only T cells present in athymic nude mice and are also found in normal animals (9). Some TCR{gamma}{delta}+ T cell populations seed (initially in some cases from the fetal or neonatal thymus) various epithelial sites such as the intestine, skin, or lung as self-renewing populations that differentiate locally, use limited TCR repertoires, and function to maintain the integrity of particular epithelia (10, 11). Extrathymic T cell populations are known to reside in the hepatic sinusoids or as intestinal intraepithelial lymphocytes (IEL). Hepatic and some intestinal IEL extrathymic T cells have a large granular morphology (12). Both populations contain autoreactive cells because they have not undergone negative selection in the thymus, but exhibit poor responses to T cell mitogens (12). Extrathymic populations have phenotypic properties of activated T cells, constitutively expressing high levels of LFA-1 and IL-2R{beta} as well as B220 (13), a unique expression of adhesion molecules and several NK markers (12).

In the present study, the development of T cell lineages and the role of the thymus as a source of immature T cells in SGs were examined using the Thy-1/CD45RC/RT6 expression model. In addition, surface phenotypes were examined to compare SG lymphocytes with other conventional and extrathymic populations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female Fischer 344 rats were purchased from Harlan Sprague Dawley (Indianapolis, IN), housed under conventional conditions, and provided with nonmedicated food and water ad libitum for at least 1 wk after receipt to allow recovery from shipping stress. Experiments were performed using young adult rats between 6 and 10 wk of age. Adult thymectomies were performed by the vendor. Thymus removal was verified by postmortem examination of a portion of each group of rats. Due to the proximity of the surgical site, the SGs and lymph nodes (LNs) were examined grossly for signs of vascularity, capsular damage, hemorrhaging, or other surgical injury upon removal for isolation of T cells (12–14 or 42 days after thymectomy). All tissues, when compared with tissues from normal rats, appeared grossly identical. All procedures conformed to the standards established by the U.S. Department of Health and Human Services.

Isolation of SG and LN mononuclear cells (MNCs)

Rats were sacrificed by CO2 narcosis, and SGs were removed and minced in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 5 mM CaCl2, 10 mM HEPES buffer, 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, and 2% FBS (HyClone, Logan, UT). MNCs were obtained by a modification of enzymatic procedures described earlier (14, 15). Briefly, the glandular fragments were dissociated with collagenase, and the released MNCs were isolated by centrifugation through a discontinuous (75, 55, and 40%) Percoll gradient. The yield of MNCs obtained using this procedure was 1 x 105 MNCs/gland for PGs and 2.2 x 105 MNCs/gland for SMGs, with viabilities >95%, as determined by trypan blue exclusion. Six or twelve rats were required to obtain sufficient MNCs from SMGs or PG, respectively, for each three-color FACS determination and the appropriate autofluorescence, single-color, and isotype controls. Pooled cervical and mesenteric LNs were minced and then dissociated by pressing through an 80-mesh stainless steel sieve. The cell suspension was passed through glass wool, then centrifuged through a Percoll gradient. Because viable SG lymphocytes could not be obtained by mechanical, nonenzymatic procedures, preliminary FACS analyses were made comparing LN MNCs obtained by collagenase digestion with those prepared by mechanical dispersion. It was found that the lymphocyte surface markers tested in these studies were not affected by the collagenase treatment.

Monoclonal Abs

The mAbs against CD3 (G4.18, biotin-, FITC-, or PE-conjugated), TCR{alpha}{beta} (R73-PE), TCR{gamma}{delta} (V65-PE), CD45R (B220, HIS24-biotin), Thy-1 (OX7-PE or -FITC), CD45RC (OX22-FITC or -biotin), CD4 (OX38-FITC), CD8{alpha} (OX8-PE), CD8{beta} (341-FITC), NKR-P1A (10/79-FITC), CD11b/c (Mac-1/CR3, OX42-PE), and CD11a (LFA-1, WT.1-PE) were obtained from BD PharMingen (San Diego, CA). Anti-CD103 ({alpha}E{beta}7, OX62-FITC) was obtained from Serotec (Raleigh, NC). The anti-RT6.2 mAb (GY1/12) was also obtained from Serotec as a culture supernatant and was purified using a Pierce protein A/G column kit (Pierce, Rockford, IL). Biotinylation was done using sulfo-N-hydroxysuccinimide biotin (Pierce), according to the supplier’s instructions. Streptavidin-conjugated Red670 (Life Technologies) was used in a separate incubation step to visualize the biotin-conjugated Abs.

Flow cytometric analysis

Cells (1 x 106/sample) were stained with a mixture of fluorochrome-labeled and biotinylated mAbs for 30 min on ice, washed, then incubated with streptavidin-conjugated Red 670 for an additional 30 min. All incubations were done in PBS with 5% BSA and 0.1% sodium azide, and all reagents were pretested individually to determine appropriate dilutions and in combinations to detect interference among the reagents. The stained cells were fixed (0.5% paraformaldehyde) and stored at 4°C in the dark until analysis the next day. Flow cytometric data were acquired using a FACScan (BD Immunocytometry Systems, San Jose, CA). For two- and three-color analyses, 20,000 events were collected and viable lymphocytes were electronically gated based on the forward and side light scatter profiles of unlabeled or CD3-labeled cells. The percentages of positive lymphocytes were determined using PCLYSYS or CellQuest software (BD Immunocytometry Systems), and the background staining obtained using isotype control Abs was subtracted.

DNA content was analyzed to determine cell cycle stages and to estimate percentages of apoptotic T cells. Lymphocytes were stained with anti-CD3-PE, washed, and fixed with 70% ethanol, then stained with 4',6'-diamino-2-phenylindole-dihydrochloride/Triton X-100 staining solution (DAPI, 1 µg/ml; Molecular Probes, Eugene, OR; in 0.1% Triton X-100 in PBS-A). Cell cycle stages of CD3+ lymphocytes were determined using PCLYSYS (BD Immunocytometry Systems) software to establish the gating parameters, including: 1) forward vs side angle light scatter to identify the lymphocyte population, 2) CD3-PE fluorescence to distinguish CD3 positive and negative populations, and 3) DAPI fluorescence width vs area to identify the diploid and apoptotic cell populations. The MODFIT software program (Verity Software House, Topsham, ME) was used to calculate percentages of CD3+ lymphocytes in G0-G1, S, and G2-M phases of the cell cycle as well as percentages of hypodiploid apoptotic cells.

Statistics

Percentages of positive cells were expressed as mean ± SEM, and the significance of differences between means was determined by the Student’s t test. Probability (p) values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MNC morphology and T cell subset distribution

Giemsa staining of cytocentrifuge preparations showed that while the majority of LN MNCs were small lymphocytes, SG cells had greater morphological diversity. Both small and large lymphocytes (some with granules) were present, as were macrophages, a few neutrophils, eosinophils, mast cells, and cellular debris (data not shown). For FACS analysis, granulocytes, dead cells, debris, and cellular aggregates were excluded using light scatter properties to gate the lymphocyte population. Using forward light scatter as an estimate of cell size and side scatter as an estimate of granularity, LN MNCs were seen as a major population of small lymphocytes (89%) with a minor subpopulation of larger, more granular cells. SG MNCs, in contrast, had lower percentages of small lymphocytes (parotid SG (PG), 77%; submandibular SG (SMG), 84%). T lymphocytes could be reliably identified by fluorescence staining for CD3, and the majority were small lymphocytes (pooled cervical and mesenteric LNs, 92%; PG, 88%; SMG, 78%).

Table IGo presents flow cytometric characterization of isolated LN and SG MNC subpopulations. Single-color FACS analyses determined that LN had 66% CD3+ T cells. PG MNCs consisted of 38% T cells, whereas only 19% of SMG MNCs expressed high levels of CD3. During the detailed characterization of T cells described below, additional mononuclear populations were identified. Two-color analyses determined that ~30% of LN and PG, but only 7% of SMG MNCs were B cells (B220+). Although few NK cells were identified in LNs (<1%) and PG (4%), these comprised the largest subpopulation of MNCs in the SMGs (37%). Small percentages of macrophages and/or dendritic cells were also identified by CD11b/c staining with mAb HIS42 (LN, <1%; PG, 6%; SMG, 5%). Interestingly, an unusual subpopulation of CD3- RT6+ MNCs was identified in the SMG that comprised 10% of the MNCs.


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  		Table I. MNC subpopulations isolated from LNs or SGs

 
Thy-1/CD45RC/RT6-defined T cell subpopulations

Expression of Thy-1, CD45RC, and RT6 has been used to classify stages of post-thymic development of rat peripheral T cells. Thy-1 marks immature T cells (and B cells), and CD45RC and RT6 are expressed by mature (Thy-1-) peripheral T cells (16). Two-color FACS analyses (Table IIGo) determined that Thy-1 was expressed by 22% of LN CD3+ T cells and significantly higher percentages of PG and SMG T cells (42 and 30%, respectively). Additional two-color experiments demonstrated that 57 and 94% of the LN T cells were positive for CD45RC and RT6, respectively. Lower percentages of PG T cells were positive for CD45RC (54%) or RT6 (77%). SMG T cells also expressed CD45RC (22%) and RT6 (73%) in statistically lower percentages than LN.


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  		Table II. T cell subpopulations isolated from LNs or SGs

 
Because T cell expression of Thy-1, CD45RC, or RT6 differed between PG and SMGs as well as in comparison with LN T cells, a set of three-color stainings was done to classify immature and mature subpopulations in these tissues. CD3-gated T cells were analyzed for labeling with a combination of two of the mAbs recognizing Thy-1, RT6.2, or CD45RC. Data in Table IIIGo represent seven phenotypically defined subsets of LN or SG T cells that were identified using these Ab combinations. Calculations were made to determine the distribution of cells in each of the developmental stages depicted in Fig. 1Go (phenotypes 1–7). This figure and calculations of relative subset frequencies are based on the model of post-thymic development introduced by Kampinga et al. (16) and have been simplified by combining the low and high intensity staining of Thy-1, RT6, and CD45RC. Other two-color FACS analyses, using the DNA-staining reagent DAPI, were done to determine the percentage of apoptotic CD3+ cells (phenotype 8).


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  		Table III. Thy-1-, RT6.2-, and CD45RC- and cell cycle-defined T cell subsetsb

 


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  		FIGURE 1. Phenotypically defined post-thymic T cell developmental stages. The frequencies of CD3+ T cell subsets in LN and SGs were calculated from the seven three-color FACS phenotypes presented in Table IIIGo. This figure and the calculation of the relative subset frequencies are based on the model of post-thymic development introduced by Kampinga et al. (16 ) and have been simplified by combining the low and high intensity staining of Thy-1, RT6, and CD45RC. An estimate of apoptotic T cells was included based on DNA staining (phenotype 8).

 
Fig. 1Go shows that 17% of LN, 38% of PG, and 29% of SMG T cells displayed an immature peripheral T cell (Thy-1+) phenotype. RT6 is expressed earlier than CD45RC; therefore, Thy-1+/RT6- T cells are assumed to be CD45RC negative. These cells are the most immature subpopulation, the RTE, and comprised 4% of LN, 8% of PG, and 6% of SMG T cells. Expression of RT6 and CD45RC begins before Thy-1 is completely lost from the T cell surface (16). LN and SG T cells each had subsets of these late thymic emigrants that were Thy-1+/RT6+ and either CD45RC positive or negative (LN, 13%; PG, 30%; and SMG, 23%). Caution must be exercised when using Thy-1 as a marker for immature rat T cells because it is also expressed by activated, proliferating mature T cells (17). We confirmed that T cells with an RTE phenotype were mainly small cells and that total CD3+ populations had only rare proliferating cells (determined by DNA content; LN, <1%; PG, 8%; SMG, 1%).

By 7 days in the periphery, thymic emigrants develop to mature, Ag-naive T cells that no longer express Thy-1 and have become CD45RC and RT6 positive (7, 18). This was the largest LN subpopulation, with 63% of T cells expressing this phenotype (Thy-1-/RT6+/CD45RC+). In contrast, only 19% of PG and 9% of SMG T cells expressed the naive phenotype. Upon Ag encounter, CD45RC and RT6 are lost, and the Thy-1-/CD45RC-/RT6- phenotype of activated T cells is expressed (17, 19). There were significant differences in the proportion of LN and SG T cells displaying an activated phenotype. Less than 1% of LN T cells were Thy-1-/CD45RC-/RT6-, but 13% of PG and 25% of SMG T cells were triple negative. In the absence of continued Ag stimulation, T cells progress to end stages of post-thymic development, consisting of two distinct subsets of resting memory T cells that reexpress either CD45RC or RT6. Type 1 expresses Thy-1-/CD45RC+/RT6-, and type 2 is Thy-1-/CD45RC-/RT6+. Although type 1 memory cells were not detected in LNs, 18% had a type 2 memory cell phenotype. SGs had slightly higher percentages of memory cells overall, compared with LNs. PG and SMG had similar proportions of type 1 memory T cells (3% each). LN and PG had 18% type 2 memory cells, whereas SMG had slightly fewer (15%). In the continued presence of Ag, activated T cells are short-lived effector cells that undergo apoptosis (20). We found that low percentages of LN (2%) and somewhat higher numbers of SG T cells were apoptotic (PG, 10%; SMG, 5%), as determined by DNA staining.

Adult thymectomy

Because Thy-1 expression is lost and CD45RC and RT6 are expressed by 7–11 days in the periphery (7, 16), age- and sex-matched rats were thymectomized (Tx) to cut off the source of fresh RTE. After 12–14 days, MNCs were prepared from LNs and SGs and analyzed by flow cytometry. This time period was chosen to allow development of a mature phenotype by any RTE released just before thymectomy. Although percentages of T cells present in LN were decreased significantly following thymectomy (normal, 67% ± 1%; Tx, 55% ± 1%: p < 0.001), the percentages found in SGs did not differ significantly. Table IIGo shows that immature (Thy-1+) T cells were strikingly decreased in LN, but not significantly altered in SGs, by thymectomy. Other than an increase in the percentage of CD45RC+ LN T cells, expression of CD45RC or RT6 was not significantly affected by thymectomy in any of the tissues.

Fig. 2Go depicts the effects of short-term adult thymectomy on the Thy-1/CD45RC/RT6-defined T cell subpopulations. Thymectomy resulted in almost total elimination of the RTE subpopulation (Thy-1+/CD45RC-) in LN (normal, 21%; Tx, <1%) accompanied by an increase in the Thy-1-/CD45RC+ mature peripheral (naive and type 1 memory) T cell subpopulations (normal, 51%; Tx, 78%) (Fig. 2GoA). No significant change in the activated population was noted. The LN Thy-1+/RT6- RTE subset was also decreased to <1% by thymectomy. The mature peripheral T cell population increased from 79% in intact rats to 93% in Tx rats, whereas the activated T cell subpopulation was not significantly changed (Fig. 2GoB).



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  		FIGURE 2. Effect of adult thymectomy on the distribution of T cell developmental subpopulations. MNCs were pooled from intact or Tx female Fischer 344 rats and were analyzed by three-color flow cytometry for expression of CD3, Thy-1, and CD45RC (A, C, and E) or for CD3, Thy-1, and RT6 (B, D, and F). Representative histograms are shown, and the numerical data summarize three separate experiments. Asterisks indicate significant differences between intact and Tx rats.

 
Thymectomy did not decrease PG MNC yield as 1 x 105 cells/gland were obtained from both normal and Tx rats. Fig. 2Go, C and D, shows that the distribution of Thy-1, CD45RC, and RT6 on PG CD3+ T cells was not significantly changed following thymectomy. Similarly, thymectomy did not significantly decrease SMG MNC yield (2 x 105 per gland for both intact and Tx rats) or the percentages of T cells (normal, 20% ± 1%; Tx, 23% ± 1%). The distribution of Thy-1, CD45RC, and RT6 on SMG CD3+ T cells was also not significantly affected by thymectomy. Fig. 2Go, E and F, shows that none of the developmental subpopulations, as defined by Thy-1 and CD45RC or RT6, were significantly changed. To rule out the possibility that Thy-1 is retained longer by glandular T cells than by those in the periphery, an additional experiment was done 42 days after thymectomy. Table IIGo shows that at 42 days Thy-1+ T cells were nearly absent in LNs, but were still present in PG and SMGs, in percentages similar to those at the 12- to 14-day time point. Furthermore, there were no changes in CD45RC or RT6 expression, except CD45RC was slightly increased in SMGs, possibly reflecting an accumulation of naive or type I memory cells.

These data show that SGs have higher percentages of T cells that express an immature phenotype, compared with LN T cells. The predominant LN subpopulation is the naive, resting T cell. In contrast, one-third of PG T cells and nearly one-half of SMG T cells have an activated/memory phenotype. The distribution of the two resting memory T cell subpopulations is similar in PG and SMG with the CD45RC-/RT6+ (type 2) subset predominant. The thymectomy experiments indicate that the immature Thy-1+ T cells found in SGs may be thymus independent.

Integrin {alpha}E{beta}7, TCR, CD4, CD8, and RT6 expression

The question of whether SG T cells have components similar to extrathymic populations residing in other tissues was studied by additional two- and three-color FACS analyses. The mucosal integrin {alpha}E{beta}7 (CD103) is present on nearly all of intestinal thymus-dependent and -independent IEL as well as 40% of intestinal lamina propria (LP) lymphocytes, but is expressed by few peripheral lymphocytes (21). This study confirms that only 2% of LN T cells coexpressed {alpha}E{beta}7 (Fig. 3GoA). Significantly greater percentages (10%) of PG T cells were {alpha}E{beta}7+, and the majority (69%) of SMG T cells expressed the {alpha}E{beta}7 integrin, as determined by mAb OX62 staining.



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  		FIGURE 3. Expression of {alpha}E{beta}7 integrin, TCR{gamma}{delta}, CD8/CD4 subpopulations, CD8 isoforms, and RT6. LN and SG T cells were analyzed by FACS for {alpha}E{beta}7 integrin (CD103; A), TCR{gamma}{delta} (B), CD8{alpha} and CD4 (C), CD8{alpha} and CD8{beta} (D), and RT6 (E). The numbers are summary data obtained from three to nine experiments and represent percentages of MNCs or percentages of CD3-gated cells. The parentheses represent calculated percentages of CD3+ T cells. Asterisks indicate significant differences between LN and SGs.

 
Although TCR{gamma}{delta}+ T cells are well represented in the circulation, they are abundantly localized in various epithelial tissues, and some have an extrathymic origin (10). Fig. 3GoB shows that 2% of LN CD3+ T cells expressed TCR{gamma}{delta}. Significantly greater percentages (4%) of PG CD3+ T cells were TCR{gamma}{delta}+, and 28% of SMG T cells expressed TCR{gamma}{delta}.

Three-color staining of CD3+ cells for CD4 and CD8 revealed that all four possible phenotypes exist in similar proportions in LNs and PGs, but with markedly different frequencies in the SMG. Fig. 3GoC shows that LN and PGs had ~60% CD4+CD8- and 30% CD4-CD8+ single-positive T cells, with 2–4% of the T cells in each tissue expressing the double-positive or double-negative phenotype. In marked contrast, SMG T cells were enriched in CD8 single-positive cells (57%) and had a smaller CD4+ subpopulation (19%). The CD4-CD8- phenotype was more frequent (15%) in SMGs, compared with LN and PG.

Both major extrathymic T cell populations (hepatic and intestinal) express the CD8{alpha}{alpha} homodimer, whereas the thymus-derived population is exclusively CD8{alpha}{beta}+ (22). Also, in the microenvironment of the small intestine, CD8{alpha}{alpha} is induced on conventional CD4+ T cells that have emigrated from the periphery (23, 24). Analysis of CD8 isoform expression (Fig. 3GoD) showed that 2% of LN, 5% of PG, and 11% of SMG T cells expressed the CD8{alpha}+{beta}- homodimer (CD8{alpha}{alpha}), indicating that these T cells are extrathymically developed.

During the thymectomy experiments detailed above, we noted, unexpectedly, that SMG MNCs had a subpopulation of CD3- cells that expressed RT6 at an intensity similar to the CD3+ cells. RT6 has been shown to be expressed by all intestinal IEL at a 10-fold greater intensity than seen on peripheral LN cells (25). Fig. 4GoE shows that 10% of SMG MNCs, but none from LNs or PGs, were CD3-/RT6+.



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  		FIGURE 4. Expression of CD8, CD4, Thy-1, and LFA-1 by LN and SG NKT cells. LN and SG MNCs were analyzed by FACS for CD3 and NKR-P1A expression (A), and CD3+/NKR-P1A+ NKT cells were analyzed for expression of CD8, CD4, Thy-1, and LFA-1 (B). Data represent two to four determinations, and asterisks denote significant differences compared with LN.

 
NKR-P1A expression

Fig. 4GoA illustrates that two distinct populations of NKR-P1A+ MNCs were identified in LNs and SGs. NKR-P1A was expressed at high levels on CD3- MNCs, especially in SMG (lower right quadrants), and at low levels on CD3+ MNCs (upper right quadrants). These subsets correspond to NK cells and NKT cells, respectively. In LNs, NK cells comprised <1% of the MNCs, whereas SGs had significantly greater percentages (PG, 4%; SMG, 37%). LN and SGs also had NKT cell subpopulations, seen in the upper right quadrants (LN, 1%; PG, 4%; SMG, 14% of MNCs). These percentages correspond to 1% of the LN T cells and significantly greater percentages of SG T lymphocytes (PG, 10%; SMG, 66%).

Fig. 4GoB presents additional three-color analyses showing that the majority of the LN and SMG NKT cells were CD8+ (79 and 82%, respectively). Significantly fewer PG NKT cells were CD8+ (49%). Lower percentages of LN and SG NKT cells were CD4+ (LN, 31%; PG, 34%; and SMG, 12%). The NKT cells, like conventional (NKR-P1A-) T cells, had immature (Thy-1+) subpopulations (LN, 24%; PG, 30%; and SMG, 28%). Furthermore, significantly more PG and SMG NKT cells displayed an activated phenotype, as revealed by expression of high levels of LFA-1 (LN, 24%; PG, 37%; SMG, 52%).

These data indicate that SG T cells are enriched in {alpha}E{beta}7+ mucosal-type T cells and in NKT cells. TCR{gamma}{delta}+ T cells are present in increased numbers in SMGs. SGs have T cell subpopulations that phenotypically resemble extrathymic populations, i.e., CD4-CD8- or CD8{alpha}{alpha}. SMGs have a subpopulation of MNCs that is CD3- and RT6+, similar to an unusual population of unknown function found in rat small intestine (26).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data demonstrate that SG T cells are composed of both thymus-dependent and thymus-independent subsets that differ from LN populations and have different developmental characteristics. The data also show that SGs contain mucosal {alpha}E{beta}7+ T cells, NKT cells, NK cells, and, in the SMGs, an IEL-like subpopulation of CD3-/RT6+ cells.

Because it had been determined that immature (Thy-1+) T cells were present in SGs (3, 4), we analyzed Thy-1, CD45RC, and RT6 expression to identify immature, naive, activated, and memory T cell subpopulations. Both PG and SMGs had greater percentages of Thy-1+ T cells than LN. When the mature (Thy-1-) T cells were characterized, striking differences were seen between SG and LN populations. Whereas the largest LN T cell subpopulation was the mature, but Ag-naive, common periperal T cells (Thy-1-/RT6+/CD45RC+), SGs had fewer naive T cells and greater percentages of activated T cells. Apart from their role in adaptive immune responses to foreign Ag, mucosal T cells have a variety of functions related to maintenance of epithelial integrity, and can be chronically activated even in the absence of microbial infection (27).

Although adult thymectomy eliminated LN Thy-1+ T cells, the percentages of PG or SMG Thy-1+ T cells were not significantly decreased. These experiments indicate that none of the PG or SMG Thy-1+ T cells are RTE, and therefore must either be generated in situ or migrate to the glands from another, unknown, extrathymic source. Although low numbers of T cells could be consistently identified in SGs following neonatal thymectomy (28), these residual cells did not support the up-regulation of salivary IgA Ab responses against Ag (29) or infection (30). Thymus-independent T cells have also been demonstrated in the blood, spleen, and LNs (31), and among the IEL (26) of adult congenitally athymic nude rats. Although the ratio of CD4+ to CD8+ cells did not differ from normal T cells, the proportions of CD45RC+ and RT6+ subsets were markedly reduced, compared with normal cells (31). In the present study, SG T cell subpopulations resembled those reported in the Rowett nude rat in that a substantial proportion was CD45RC- in SMG and decreased percentages of T cells were RT6+ in both glands, compared with LN T cells.

Based on CD4 and CD8 expression patterns, SG T cell populations differed from extrathymic intestinal and liver populations. Extrathymically developed T cells in the intestine are mainly CD8+ or CD4+/CD8+ (12, 25, 27, 32), whereas those in the liver are either CD4+ or CD4-/CD8- (12). The majority of PG T cells were CD4 or CD8 single positive, and only small percentages were CD4+/CD8+ or CD4-/CD8-. Similar to intestinal IEL, most SMG T cells were CD8+ and contained a substantial subpopulation of CD4-/CD8- double-negative T cells. In the intestinal IEL compartment, the majority of double-negative cells are TCR{gamma}{delta}+ (12, 27, 33, 34). In PG, the large numbers of CD4+ T cells and low numbers of double-positive or double-negative cells resembled the intestinal LP population (32) more closely than the IEL population. Immunohistochemical studies have demonstrated that CD4+ and CD8+ T cells, B cells, and plasma cells are located in salivary interstitium and in small periductal aggregates, whereas the acini contain intraepithelial CD8+ cells (35). In contrast, the predominance of CD8+ cells, accompanied by a double-negative population in SMG, more closely resembles the intestinal IEL population (27).

Thymus-derived T cells express the {alpha}{beta}-chain heterodimeric form of CD8, whereas extrathymic T cells in both liver and intestine display the CD8{alpha}{alpha} homodimer (12), although, in some circumstances, CD8{alpha}{alpha} is expressed by activated conventional CD4+ T cells (36). We found that both PGs and SMGs T cells expressed CD8{alpha}{alpha}, but in much lower numbers than reported in the intestine (22). Thus, when CD4 and CD8 expression is considered, the majority of rat SG T cells are phenotypically similar to conventional T cells, but small percentages (5% and 11% in PG and SMG, respectively) have an extrathymic phenotype.

TCR{gamma}{delta}+ cells are abundant in epithelial tissues, have thymus-dependent and extrathymic origins, have a unique capacity to protect the host against specific pathogens, and have a broad spectrum of reactivities. They are important in immune surveillance of various epithelia and respond to distressed epithelial cells by producing growth factors that facilitate repair. Through their production of lymphokines and chemokines, TCR{gamma}{delta}+ T cells have critical regulatory roles in both adaptive and innate mucosal immune responses (10, 37). Although only small percentages of PG T cells were TCR{gamma}{delta}+, SMGs had percentages (28%) comparable with percentages (25%) reported for mouse SGs (38). The enhanced numbers of TCR{gamma}{delta}+ T cells are unique to SMGs because PGs and lacrimal glands4 have only small numbers (7%) of these cells. Although it is noteworthy that percentages of activated T cells and of TCR{gamma}{delta}+ cells are nearly identical in the SMG, further studies are required to learn whether these populations overlap and what specific role(s) SMG TCR{gamma}{delta}+ cells play in protection of the oral compartment.

The integrin {alpha}E{beta}7 (CD103) is expressed by most or all TCR{gamma}{delta}+ and some TCR{alpha}{beta}+ IEL and LP lymphocytes in various epithelia (21) and in human (39) and rat lacrimal glands.4 Furthermore, {alpha}E{beta}7 is expressed by veiled cells in lymph and by rare cells with a dendritic morphology in the spleen and LNs (21, 40). The {alpha}E{beta}7 integrin functions as an adhesion molecule whose ligand is E-cadherin on epithelial cells and has a role in retention and/or function of IEL within the epithelium (41). Integrin {alpha}E{beta}7 can be induced on peripheral T cells by TGF-{beta}, but does not function as a homing receptor (42). The majority (69%) of SMG T cells were {alpha}E{beta}7+, whereas only 10% of PG T cells expressed this marker. Because these percentages exceed the TCR{gamma}{delta}+ T cells, {alpha}E{beta}7 must also be expressed by SG TCR{alpha}{beta}+ lymphocytes.

Unexpectedly, an unusual subpopulation of CD3-/RT6+ MNCs was identified in SMG that was undetectable in PGs or LNs. Similar cells have been reported in rat small intestine, in which RT6 is expressed by 99% of IEL at about a 10-fold higher density than on peripheral T cells (25). In athymic nude rats, all IEL are RT6+, but only half express CD3+ (25). In contrast to peripheral T cells in which the occurrence of RT6 is a late differentiation step, it appears that immature T cell precursors, developing locally, express RT6 before the CD3 TCR complex in the intestinal environment (26, 43). Although it can be speculated that the CD3-/RT6+ cells in SMG represent immature locally developing T cells, further studies are needed to determine whether the SMG environment has a capacity, similar to the intestine (44, 45), for local production of T cells. Similar CD3-/RT6+ cells have been identified in lacrimal glands in low percentages (1%, unpublished observation).

CD1-restricted NKT cells are distinct from both conventional T cells and NK cells, represent a major subpopulation of lymphocytes in thymus, spleen, bone marrow, and liver, but are rare in LNs. They express both TCR and NK cell surface markers. Following TCR-mediated activation, NKT cells produce large amounts of IL-4 and IFN-{gamma}, similar to T cells, and subsequent to NKR-P1-mediated activation, they have cytolytic activity similar to NK cells. NKT cells have immunoregulatory roles in vivo that are distinct from conventional T cells. They participate in removing mycobacterial pathogens, contribute to tumor rejection, provide help to B cells, and appear to be required to prevent autoimmunity (34). There have been conflicting reports concerning whether NKT cells in the periphery arise in the thymus or have an extrathymic origin, but Coles and Raulet (46) present strong evidence that liver CD1-restricted NK1.1+ T cells in mice arise in the thymus but expand in the liver.

Our studies show that NKT cells are the predominant (66%) T cell population in rat SMGs and are also present in PGs (10%) as well as in lacrimal glands (34%).4 The majority of glandular NKT cells are CD8+, although small numbers of CD4+ cells are also present. Others have reported that the majority of rat NKT cells are CD8+ (47). Along with the canonical CD1-restricted NKT cell population, which in humans and mice is CD4+ or CD4/CD8 double negative, NK markers are expressed on some activated and memory conventional CD8+ and CD4+ T cells (48, 49, 50, 51, 52). Our data show that 50% of SG NKT cells were LFA-1high, suggestive of activated cells. Further phenotypic and functional investigations are required to determine whether SG NKT cells represent canonical, CD1-restricted NKT cells, activated/memory conventional T cells, or subpopulations of both.

Interestingly, an abundance of NKT cells has been reported (53) in the SMG of aly/aly mutant mice with Sjögren’s syndrome, and these authors concluded that the NKT cells in the SGs were extrathymically generated in situ. A percentage of rat SG NKT cells were immature Thy-1+ cells, suggesting either in situ generation or recent emigration from some other generative site. Although mouse NKT cells in the liver originate in the thymus from slowly proliferating precursors, the intrahepatic NKT cells have a greatly increased proliferation rate. Overall, NKT cell numbers did not increase in the liver, suggesting that this population either has a high death rate, migrates out of the liver, or changes its phenotype (46). Further investigation is required to determine whether liver NKT cells can migrate to SGs or other mucosal sites.

NK cells form a third group of lymphocytes that are distinct from T and B cells and, via their cytotoxicity, are important as a first line of defense against infectious agents and metastatic cells (54). In addition to being cytotoxic, NK cells produce a variety of cytokines and chemokines that are important in regulating normal and autoimmune T and B cell responses (55, 56), in regulating hemopoiesis, in recruiting leukocytes to the site of inflammation, and in blocking fetal-maternal immune responses (57). The large percentage of NK cells in SMGs and the small population in PGs is an interesting focus for future investigations of innate and acquired salivary immune responses as well as the physiology of these tissues.

These phenotypic studies identify unique MNC populations in SGs and provide an important foundation for further investigation into the cellular basis for regionalization of the mucosal immune system. These distinctions must be considered when designing effective immunotherapy to protect particular mucosae or intervene in immunopathological processes occurring in mucosa-associated tissues.


   Acknowledgments
 
We thank Drs. Robert Swanborg and Randall Gill for their helpful comments on this manuscript, and the assistance in flow cytometry by Eric Van Buren is gratefully acknowledged.


   Footnotes
 
1 This work was supported by Grant DE-09658 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Nancy L. O’Sullivan, Department of Immunology and Microbiology, Wayne State University School ofMedicine, 540 East Canfield Avenue, Detroit, MI 48201. Back

3 Abbreviations used in this paper: SG, salivary gland; LN, lymph node; RTE, recent thymic emigrant; PG, parotid SG; SMG, submandibular SG; MNC, mononuclear cell; Tx, thymectomized; IEL, intraepithelial lymphocyte; LP, lamina propria; DAPI, 4',6'-diamino-2-phenylindole-dihydrochloride. Back

4 N. L. O’Sullivan, C. A. Skandera, and P. C. Montgomery. Development of T cell lineages in rat lacrimal glands. Submitted for publication. Back

Received for publication August 15, 2000. Accepted for publication February 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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