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Originally published online as doi:10.1189/0507279 on September 7, 2007

Published online before print September 7, 2007
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(Journal of Leukocyte Biology. 2007;82:1430-1436.)
© 2007 by Society for Leukocyte Biology

Higher maturity and connective tissue association distinguish resident from recently generated human tonsil plasma cells

Francisco Medina, Carmen Segundo, Gema Jiménez-Gómez, Inés González-García, Antonio Campos-Caro and José A. Brieva1

Servicio de Inmunología and Unidad de Investigación, Hospital Universitario Puerta del Mar, Cádiz, Spain

1Correspondence: Servicio de Inmunología, Hospital Universitario Puerta del Mar, Avenida Ana de Viya 21, 11009 Cádiz, Spain. E-mail: josea.brieva.sspa{at}juntadeandalucia.es


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ABSTRACT
 
Human plasma cells (PC) are present in cell suspensions obtained from the tonsil by mechanical disaggregation (PCMECH). The present study shows that a collagenase treatment of tonsillar debris remaining after mechanical disaggregation yielded similar proportions of PC (PCCOLL). Moreover, PCMECH were present in suspensions highly enriched in germinal center cells whereas PCCOLL contained most of the IgA-secreting cells, suggesting their predominant location in follicular and parafollicular areas and connective tissue-rich zones such as tonsil subepithelium, respectively. Tonsil PCMECH and PCCOLL shared the phenotype CD38high CD19+ CD20low CD45high, expressed equivalent amounts of PRDI BF1/Blimp-1 transcription factor, and carried similarly mutated IgVH6 genes. However, they differed in several features. 1) PCMECH still expressed the early B cell transcription factor BSAP and were HLA-DRhigh; in contrast, PCCOLL were BSAP–and HLA-DRlow. 2) PCMECH were CD95+ and Bcl-2+/– whereas PCCOLL showed CD95+/– and Bcl-2+ expression; in addition, PCMECH exhibited increased spontaneous apoptosis. 3) The two PC subsets exhibited distinctive adhesion molecule profiles, since PCCOLL expressed higher levels of CD31, CD44, and CD49d, but a lower level of CD11a than PCMECH. These results suggest that PCMECH are recently generated, short-living PC, and PCCOLL constitutes a subset with higher maturity and survival, which resides in connective tissue-rich areas.

Key Words: PC biology • adhesion molecule • B cell transcription factor • differentiation


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INTRODUCTION
 
Plasma cells (PC) are the final effector stage of B cell differentiation; as such, they become cellular factories specialized in Ab production. Experiments in rodent models have demonstrated that, upon Ag immunization, PC are first detected in parafollicular areas and, slightly later, in the germinal centers (GC) of Ag-draining peripheral lymphoid tissues [1 2 3 ]. Although most of these recently generated PC are short-living cells that die rapidly by apoptosis in these inductive territories [4 ], some acquire migratory capability [5 , 6 ], enter the circulation, and home in on distant deposit areas such as the bone marrow (BM) and the mucosal lamina propria (LP); once in these locations, they comprise the pool of resident long-living mature PC and are responsible for serum and mucosal Ig levels, respectively [6 7 8 9 10 11 12 ].

Recently generated PC from human peripheral lymphoid organs and blood exhibit distinctive characteristics when compared with resident PC from deposit organs (BM, LP): 1) they produce less Ig in culture [13 ]; 2) they are prone to undergo apoptosis [14 , 15 ]; and 3) they show a less mature phenotype in terms of differentiation markers and display an organ-specific adhesion molecule profile [16 ]. Taken together, these data indicate the existence of a PC maturation gradient from inductive peripheral tissues to final deposit organs. Nevertheless, mice models have provided evidence of the occurrence of a certain population of resident long-living PC apparently remaining in the spleen [17 ]. In humans, the presence of an equivalent splenic PC population has also been suggested [18 ].

The human palatine tonsil is a systemic secondary lymphoid organ that, through the broad subepithelial crypt zone, is connected with the highly septic oral cavity; accordingly, this organ plays a relevant role in the defense against infection by local pathogens [19 ]. Since tonsil is the bona fide experimental model of human secondary lymphoid organ, it could be suitable for examining the possible presence of such resident peripheral PC compartment. PC located in tonsil sites surrounded by abundant connective tissue, such as the subepithelium and certain perivascular areas [20 21 22 ], are probably neglected during the mechanical disaggregation procedure usually employed for tonsil cell isolation, and they could belong to this putative subpopulation of resident PC. In the present study, tonsillar debris, usually discarded after mechanical disaggregation, was further digested with collagenase. This treatment released an additional PC subset that seems to be intermediate in terms of functional and phenotypic features between tonsil PC obtained by simple mechanical means and BM PC, and might represent a tonsillar resident PC subpopulation.


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MATERIALS AND METHODS
 
Materials
Cycloheximide and collagenase-V were purchased from Sigma (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC) -labeled monoclonal antibody (mAb) against CD11a, CD20, CD44, and CD45, phycoerythrin (PE)-labeled mAb against CD19, CD20, CD31, CD49d, CD95, and HLA-DR, FITC-annexin V, and the corresponding isotypic negative controls were provided by Becton Dickinson (San Jose, CA, USA). Cy-chrome (CyC)-labeled mAb against CD38, the corresponding negative control, were from Caltag (San Francisco, CA, USA). EDTA was purchased from Pharmacia Biotech (Uppsala, Sweden). Purified mAb against CD31 was provided by Coulter (Miami, FL, USA). FITC-conjugated mAb against bcl-2 and p63 (VS38c clone), FITC-conjugated rabbit anti-human {gamma}, {alpha}, and µ Ig heavy chains, and an intrastain kit were purchased from Dako (Glostrup, Denmark). Goat anti-mouse IgG magnetic microbeads, selection columns of MS+ type, and a miniMACS magnet were obtained from Miltenyi Biotec (Auburn, CA, USA). Flat-bottom (96-well) culture plates and U96 Maxisorp plates used for ELISA were provided by Nunc (Roskilde, Denmark). Unconjugated and peroxidase-conjugated goat F(ab)'2 anti-human IgG, IgA, and IgM, used in ELISA, were purchased from Biosource (Camarillo, CA, USA). Rhodamine-conjugated polyclonal goat antibody against human IgA used in immunocytochemical detection of IgA+ PC was from Jackson Immunoresearch (West Grove, PA, USA).

Tonsil B cell isolation
Tonsils were obtained from subjects undergoing tonsillectomy for chronic tonsillitis. Approval for this study was obtained from the institutional review board (Comisión Ética, Hospital Universitario Puerta del Mar) according to the Declaration of Helsinki. The tonsillar tissue was placed on a Petri dish containing HBSS and slashed with a couple of scalpels until a mass of fibrous residue of the organ was produced and the surrounding medium turbidity decreased. This mechanical disaggregation step yielded a cell suspension that was stored at 4°C. The remaining tonsillar tissue was washed several times in HBSS and used in an additional enzymatic digestion step with collagenase-V (1 mg/ml in RPMI-1640, 15 min, at 37°C in a shaking bath). The subsequent cell suspension was also harvested. The two cell suspensions were washed in culture medium and separated into T and non-T cell populations by a previously reported rosette technique [23 ]. Non-T cell populations consisted mostly of B cells (>97% CD19+ cells) and were termed BMECH (after mechanical disaggregation) and BCOLL (after collagenase digestion) cell fractions. CD31+ B cells were purified from BMECH and BCOLL cell fractions by positive immunomagnetic selection, as described [24 ], and used in phenotype and isolation FACS assays. This selection step was used because CD31 expression by PC is distinctively high, and so allowed for the pre-enrichment of these cells.

Immunohistochemical detection of tonsil IgA+ plasma cells
Tonsil samples were frozen in cooled (–70°C) isopentane and embedded in OCT Tissue-Tek. Consecutive 10 µm cri-sections were obtained and stained with either conventional hematoxylin/eosin or rhodamine-conjugated polyclonal antibody against human IgA. In brief, sections for immunofluorescence studies were fixed in 4% paraformaldehyde, permeabilized in 0,1% triton, blocked with 1% goat serum, and stained with the anti-IgA antibody (1:200 dilution). After every step, samples were washed extensively in PBS. Both kinds of preparations were explored by light and conventional fluorescent microscopy (Olympus BX40. Hamburg, Germany), and equivalent fields in the consecutive sections were identified and photographed (Olympus DP71 camera, Olympus Cell D software).

Cell culture and Ig ELISA
BMECH and BCOLL cells were adjusted to 106 cells/ml in a culture medium consisting of RPMI 1640 supplemented with 10% FCS, L-glutamine (10 mM), and gentamycin (0.05 mg/ml), then cultured in 96-well plates in a final volume of 250 µl/well at 37°C with 5% of CO2. In some experiments, cells were collected after 18 h and the proportion of apoptotic PC was determined. After 4 days, cell-free supernatants (SN) were collected and IgG, IgA, and IgM secretion was tested by ELISA in microtiter plates, as reported earlier [25 ].

Flow cytometry
Two- and three-color labeling experiments were performed as reported [16 ]. In brief, 200 µl of BMECH and BCOLL cell fractions (at 0.5–5x106 cell/ml) were incubated with optimal concentrations of mAb for 20 min in the dark at 4°C, followed by two washes. PC were identified as cells exhibiting low CD20 and high CD38 expression (CD20low CD38high) [26 ]. The color combination used for this detection was CyC-CD38 and either PE- or FITC-CD20, using the free channel for the molecule under study. Apoptotic PC were assessed using the annexin-V binding assay, as described previously [14 ]. Intracytoplasmic Ig-containing cells were determined in permeabilized PC, as reported [16 ]. FACS analysis was performed on a FACScalibur cytometer (Becton Dickinson) equipped with an air-cooled argon ion laser emitting 15 mW at 488 nm. The instrument was equipped with three fluorescence detector photomultiplier tubes and a standard filter set (530/30 nm band pass for FITC, 585/42 nm band pass for PE, and 650 nm long pass for CyC). Cell analysis was performed with CELLQUEST software (Becton Dickinson).

Plasma cell isolation and identification
Sorting of CD38high cells from both BCD31+ cell fractions was performed on a FACStar-Plus cytometer (Becton Dickinson) equipped with an air-cooled argon ion laser emitting 100 mW at 488 nm. Cells were previously labeled for CD20 (FITC) and CD38 (PE), and CD38high cells were gated with LYSSIS-II software (Becton Dickinson) and sorted in C-normal mode, as reported [16 ]. Isolated CD38high cells (0.1 106 cells in 100 µl PBS) were cyto-centrifuged on slides and stained by the Giemsa technique. Percentage of cells with PC morphology was determined by optical microscopy.

Detection of the B cell transcription factors Blimp-1 and BSAP
The presence of transcripts of Blimp-1 and BSAP was investigated in purified PCMECH and PCCOLL by reverse transcriptase-polymerase chain reaction (RT-PCR). To this end, total RNA from each cellular fraction was purified using the High Pure RNA Isolation Kit (Roche, Barcelona, Spain) and cDNA was synthesized using random hexamers with the Transcriptor First Strand cDNA Synthesis Kit. Then PCR was performed with the following oligonucleotide primers: for Blimp-1 sense primer 5'-ATGCGGATATGACTCTGTGGA-3' and anti-sense primer 5'-CTCGGTTGCTTTAGACTGCTC-3', and for BSAP (sense) 5'-CAGCATAGTGTCCACTGGCT-3' and (anti-sense) 5'CCTGTCAGCGTCGGTGCTGA-3'. Three microliters of cDNA were amplified in a PTC-100 MJ Thermocycler (MJ-Research Inc., Waltham, MA, USA) using each primer and Taq DNA polymerase (Biotaq, Bioline Ltd., UK). The amplified products were analyzed on a 1.2% agarose gel containing ethidium bromide and visualized by UV light illumination. The quantity of β-actin cDNA was evaluated by using a sense primer 5'-TACCACTGGCATCGTGATGGACT-3' and anti-sense primer 5'-CGTCACACTTCATGATGGAG-3' as cDNA internal control.

Sequence analysis of IgVH6 gene
The IgVH6 gene family was amplified from cDNA (synthesized above) using the Expand High Fidelity Plus PCR System (Roche, Barcelona, Spain). Briefly, the strategy used was as follows: a common forward specific primer for VH6 signal peptide (5'-ccggatccGGCCTCCCATGGGGTGTCCTG-3') was used in conjunction with reverse isotype primer specific for either C{gamma} (5'-ccggatccGAAGACCSGATGGGCCCTTGGT-3'), Cµ (5'-ccggatccGAAAAGGGTTGGGGCGGATGC-3'), or C{alpha} (5'-ccggatccGAAGACCTTGGGGCTGGTCGG-3'). A tail sequence (lowercase letters) containing a restriction site for BamHI endonuclease was added in all primers used. Amplified PCR products were cloned after BamHI digestion into the BamHI linearized pBluescript plasmid (Stratagene, La Jolla, CA, USA). Positive clones were sequenced using the Big Dye V3.1 Terminator Kit and the ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Nucleotide sequences were analyzed and compared with the IMGT IgVH database (http://imgt.cines.fr/).

Statistical analysis
Results are expressed as the mean and SE. Differences were analyzed by the Student’s t test.


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RESULTS
 
Tonsillar PC subsets released by mechanical and enzymatic purification methods; in vitro Ig secretion capability
After mechanical disaggregation, the released cells were removed and collected, and the remaining tonsillar tissue was exhaustively washed and further treated with collagenase to test whether additional PC could be recovered. This digestion step also yielded a cell suspension, and mechanically and enzymatically released cellular populations were T cell depleted to generate B cell fractions (termed BMECH and BCOLL, respectively). Figure 1A shows a representative FACS analysis of CD20/CD38 staining of BMECH (left dot plot) and BCOLL (right dot plot) cell fractions. Both methods released an equivalent percentage of PC, determined as CD38high cells (1.67%±0.3% vs. 1.64%±0.3%, mean±SE, n=12). The BMECH cell fraction was enriched in germinal center (GC) B cells (CD20high CD38+ cells) [27 ] with respect to the BCOLL cell fraction, as the former contained 4-fold more GC. B cells (Fig. 1B ; 41.3%±8% vs. 11.7%±3.7%; mean±SE; P<0.05). The capacity of in vitro spontaneous Ig production, a feature of PC, was next explored in both B cell populations. As can be seen in Fig. 1C , BCOLL cells secreted twice as much IgG and four times as much IgA as BMECH cells, despite the proportion of CD38high PC being similar in both cell preparations. The proportion of intra-cytoplasmic IgA+ CD38high cells was also four times higher in BCOLL than in BMECH (12.8% ± 2 vs. 3.1% ± 0.3, mean ±SE; n=4; P<0.03). Immunohistochemical studies of tonsil sections revealed that IgA+ PC were almost totally excluded from the follicles, and clearly enriched in subepithelial areas (Fig. 1D) .


Figure 1
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  		Figure 1. Human tonsil B cells isolated by mechanical disaggregation (BMECH) or collagenase treatment (BCOLL): B cell distribution and spontaneous Ig secretion capability. (A) Dot plot histograms showing representative CD20/CD38 staining of tonsil BMECH and BCOLL cells. PC are determined as a CD38high cell subpopulation (labeled PC), GC B cells were defined as CD20high CD38+ cells (labeled GC), and other small B lymphocytes were CD20+ CD38– (labeled SBL). (B) Distribution of tonsil B lymphocyte subsets in BMECH and BCOLL cell fractions. Results are represented as the mean percentage ± SE (n=4). (C) BCOLL and BMECH cell spontaneous Ig secretion was assessed by measuring Ig in the culture supernatants obtained after 4 days. Results represent the mean ±SE of 5 experiments. Statistical significant difference (P<0.05) is denoted by an asterisk (*). (D) Localization of tonsil IgA+ PC: Consecutive tonsil sections were stained with hematoxylin and eosin (left microphotograph) or with a rhodamine-conjugated polyclonal antibody against human IgA (middle microphotograph). GC and epithelium (E) are marked with dotted lines. A representative image of the middle photograph is magnified (x40) on the right-hand microphotograph.

Expression of Blimp-1 and BSAP and IgV-H6 gene mutations in isolated tonsillar PCMECH and PCCOLL populations
To examine additional features of the two tonsil PC subsets under study, they were purified as CD38high cells in a two step isolation protocol, as reported [16 ]. Figure 2A shows a representative CD20/CD38 staining of PC isolated by this procedure from the BMECH (left dot plot) and from the BCOLL cell fraction (right dot plot). Highly purified CD38high populations (>95%) were obtained from both fractions and further confirmed as PC (PCMECH and PCCOLL) by Giemsa staining, as can be seen in Fig. 2A (photograph inserts). Purified PC mRNA was obtained, and cDNA was generated to determine transcription factor expression by RT-PCR and mutation frequency in Ig-VH6 gene by cloning and sequencing. Figure 2B shows an agarose gel of three independent experiments comparing PCMECH and PCCOLL expression of BSAP, a transcription factor down-regulated in PC [28 , 29 ] and Blimp-1 (PDRI-BF1), a well-established master regulator of the PC maturation program [30 ]. BSAP was negative in PCCOLL but positive in PCMECH (upper panel), whereas Blimp-1 was equally positive in both tonsillar PC populations (middle panel). Figure 2B (lower panel) shows densitometric measurement of the obtained bands expressed as BSAP/BLIMP-1 ratio, confirming the data mentioned above.


Figure 2
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  		Figure 2. Purification of PC subsets from BCOLL and BMECH: Blimp-1 and BSAP expression and IgVH6 gene mutations. (A) Dot plot histograms showing a representative CD20/CD38 staining of PCMECH and PCCOLL purified after a two-step isolation protocol. Giemsa cell staining is shown in the corresponding micrograph on the right. (B) BSAP and Blimp-1 mRNA expression in purified PCMECH and PCCOLL determined by RT-PCR using an equal amount of cDNA. PCR products were quantified using the Quantity One software (Bio-Rad, Hercules, CA, USA) and corrected by subtracting the local background. Data were expressed as the ratio of BSAP/BLIMP-1 expression in three independent experiments (numbered 1–3, m = PCMECH, c = PCCOLL, and mw = molecular weight ladder). (C) Bar histograms represent the number of total mutations observed in the IgVH6 gene sequenced from purified PCMECH (open bars) and PCCOLL (gray bars). Results are expressed as the mean ±SE of 34 and 51 (IgM), 67 and 62 (IgG), and 63 and 49 (IgA) sequences for PCMECH and PCCOLL, respectively (GenBank accession # EF561294-EF561619).

To examine the Ig gene mutational status in PCMECH and PCCOLL, sequences from Ig-VH6 gene were amplified for the three most frequent isotypes (IgM, IgG, and IgA) and their sequences were analyzed. Figure 2C shows the number of total mutations for the three isotypes in the whole sequence. Both PC subsets showed mutated Ig genes without any apparent difference between PCMECH and PCCOLL in total mutation numbers (7.02±0.71 vs. 9.08±1.53 for IgM, 14.54±1.62 vs. 13.25±1.44 for IgA, 9.90±0.95 vs. 10.76±0.90 for IgG; not significant). No differences were observed when replacement mutations were quantified in either the whole sequence or the CDR1+2 region, although (as expected) R mutations were more frequent in the CDR1+2 than in the framework region (data not shown). In these analyses, IgA was the isotype with the highest number of mutations, IgM had the lowest, and IgG had intermediate numbers; all these differences were significant (data not shown).

Differentiation, survival, and adhesion molecule expression in tonsil PCMECH and PCCOLL
Taking advantage of the correspondence between CD38high phenotype and PC stage, the expression of additional maturational and survival markers by PCMECH and PCCOLL was analyzed by FACS in the corresponding B cell fractions. Figure 3A shows a representative example of this comparative study. Results summarizing the percentage and MFI of positive PC in several experiments are also included (Fig. 3B and 3C , respectively). Some differentiation markers, such as CD19, CD20, and CD45, were present equally in both tonsillar PC populations. In contrast, HLA-DR showed a 2-fold increase in expression level in PCMECH with respect to PCCOLL. Survival-related molecules exhibited an opposite distribution in the two PC populations: whereas PCMECH expressed the death receptor CD95 but very little of the apoptosis protection factor Bcl-2, PCCOLL barely expressed CD95 but stained prominently for Bcl-2. Spontaneous apoptosis by PCMECH and PCCOLL was determined as annexin-FITC+ CD38high cells in each population after 18 h of culture. The results of two independent experiments revealed that apoptotic PC were more frequent in PCMECH than in PCCOLL (25% and 32% vs. 6% and 10%, for PCMECH and PCCOLL, respectively).


Figure 3
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  		Figure 3. Comparative phenotype of tonsil PCCOLL and PCMECH. (A) Representative example of the PCCOLL and PCMECH histogram expression of several differentiation (HLA-DR, CD19, CD45), survival (CD95, Bcl-2), and adhesion (CD49d, CD44, CD31, CD11a) molecules. The summaries of five different experiments are expressed as the mean ±SE of the percentage (B) and the MFI (C) of expression of the indicated molecules. Statistical significant difference (P<0.05) is denoted by an asterisk (*).

Finally, using the same FACS analysis strategy, adhesion molecule expression was examined as further potential evidence of heterogeneity between both tonsil PC populations. Figure 3A shows a representative example of this comparative study. Results representing the percentage and MFI of positive PC in several experiments are also summarized (Fig. 3B and 3C , respectively). CD49d, CD44, and CD31 were expressed more intensely in PCCOLL than in PCMECH (3-, 2-, and 2-fold more intensely, respectively). Conversely, the expression of the {alpha}-integrin CD11a was clearly higher in PCMECH than in PCCOLL. Several other molecules were similarly expressed by most of the cells from both tonsil PC subsets, including CD9, CD21, CD22, CD27, CD40, CD50, CD54, and intracellular VS38c (data not shown). Collagenase treatment of BMECH cell fractions did not modify the phenotype of PCMECH.


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DISCUSSION
 
The human palatine tonsil contains areas where lymphocyte are loosely juxtaposed (follicles and parafollicular areas) [31 ], from which cells can be easily removed by simple mechanical disaggregation, and other compartments rich in connective tissue [32 ], where lymphoid cells are surrounded by dense extracellular matrix networks, thus requiring collagenase digestion to be released. Present results confirm this notion, as the BMECH cell fraction was clearly enriched in GC B cells (a loose cellular compartment) compared with the BCOLL cell fraction. It is reasonable to expect that tonsil PC would follow a similar pattern of segregation: PC localized in GC and parafollicular areas [33 ] would be preferentially collected in the BMECH cell fraction, whereas PC associated with areas rich in connective tissue, such as the subepithelium [20 , 33 ] and certain perivascular areas [21 , 22 ], would be enriched in the BCOLL cell fraction. The prominent IgA production in vitro, as well as the parallel intracytoplasmic IgA+ PC enrichment showed by PCCOLL, confirms the idea that collagenase treatment leads to the release of IgA-secreting PC from the subepithelial area, where these cells preferentially accumulate in vivo [20 , 33 ]. In addition, cultured PC present in BMECH and BCOLL cells differed considerably in their capacity for Ig production even though both cell fractions contained equivalent PC percentages. Thus, PCCOLL spontaneously secreted twice as much IgG and 4-fold as much IgA as PCMECH, indicating an overall differential Ig production capacity.

Further results reveal that human tonsil PCMECH and PCCOLL exhibited the same CD19+ CD20low CD45high phenotype, showed a similar quantity of the PC master regulator PRDI-BF1/Blimp-1, and bore a similar frequency of mutated Ig-VH6 genes, strongly suggesting that both PC subsets are mostly generated from post-GC cellular precursors. Despite these similarities, they differed in several PC maturational characteristics. First, PCMECH still expressed BSAP, a transcription factor relevant for B lymphocytes at all earlier differentiation stages but absent in mature PC [28 , 29 ]. In fact, the BSAP expression by PCMECH is probably residual, as this factor inhibits the expression of Blimp-1 and the subsequent progression into the PC maturation program [34 ]. The expression of BSAP and CD20 by tonsil PCMECH suggests that this subset might be a normal counterpart of a recently described clinical subtype of multiple myeloma in which malignant PC exhibited this same phenotype [35 ]. PCMECH also showed high expression of HLA-DR, a molecule that is down-regulated early in the PC maturation program [36 ], as demonstrated in more mature BM and LP PC [16 , 37 ]. Moreover, PCMECH also expressed the death receptor CD95 but hardly any of the apoptosis protection factor Bcl-2; accordingly, they exhibited an increased tendency to undergo spontaneous apoptosis. The enhanced spontaneous apoptosis showed by PCMECH may help explain their lower Ig secretion capacity. These data suggest that PCMECH are recently generated PC that probably have only a short survival capacity. On the other hand, PCCOLL did not contain BSAP; they expressed a low level of HLA-DR, were almost negative for CD95 but positive for Bcl-2 staining, and exhibited enhanced resistance to apoptosis. These properties indicate that PCCOLL are at a more advanced stage of maturation and likely have a longer life span in vivo.

A comparison of the adhesion molecule profiles showed by PCMECH and PCCOLL provides additional evidence of heterogeneity. Molecules such as CD49d and CD44 were more intensely expressed in PCCOLL than in PCMECH, in probable correlation with their attachment capacity to connective tissue-rich locations, where their respective ligands are especially abundant [38 ]. Moreover, it has been demonstrated that CD49d interaction with its ligands is necessary for the survival of resident human and murine BM PC [39 , 40 ]; accordingly, adhesion by means of this integrin could have a similar function in PCCOLL. Taken together, the predominant pattern of CD49d, CD31, and CD44 adhesion molecules observed on PCCOLL is identical to that described for human BM PC, the only difference being that the intensity of expression is even higher in the latter cells [16 ]. These observations, combined with those discussed above, support the view that PCCOLL might play a role as a tonsil resident PC subset located in persistent PC lodging niches.

Unlike the case of CD49d, CD44, and CD31, the {alpha}-integrin CD11a was more intensely expressed on PCMECH, a feature that might be related to their differential egress potential. The notion that recently generated PC can exit the tonsil is based on the observation that, after intra-tonsillar Ag delivery, Ab-secreting PC can be detected in the circulation [41 ], reaching distant locations such as bronchial associated lymphoid tissue [42 ] and BM [43 ]. PC of mice defective for CD18 (the β subunit pair of CD11a) accumulate in the medullary cords, unable to egress the lymph node via the lymphatic vessels in order to enter the circulation, which indicates that this integrin has a role in the PC exit process [44 ]. Therefore, it is tempting to speculate that tonsil PC recently generated in the follicle and parafollicular areas, and which have acquired migratory capacity, are contained in the CD11a+ PCMECH fraction, and that they could leave the tonsil by a CD18-dependent mechanism. The other CD18 {alpha}-integrin partners, CD11b and CD11c, were negative in both tonsil PC subsets (data not shown).

In summary, the present study reveals that human tonsil contains two PC subsets that differ in several relevant aspects. PCMECH, present in the follicular and parafollicular areas, are less mature, suggesting they are recently generated PC, many of which will die by apoptosis in a few days, as reported earlier for this kind of PC in mice [3 , 4 ] and humans [14 , 15 ]. Some PC contained in this fraction might potentially egress from tonsils by a CD11a/CD18-dependent mechanism and colonize distant sites. On the other hand, PCCOLL exhibit more mature differentiation features, longer survival, and higher Ig secretion. Furthermore, the high CD49d, CD44, and CD31 expression observed on PCCOLL could contribute to their anchorage in close proximity to undetermined stromal or auxiliary cells, which in turn would constitute lodging niches to maintain them in a long-living resident status, as suggested for certain splenic PC niches [17 , 18 ]. PCCOLL appear to be similar to long-living PC recently reported in human tonsil samples grafted into SCID mice [45 ]. The origin of tonsil PCCOLL remains unknown. PCCOLL could originate from recently generated PC in the same organ, as indicated by intra-tonsillar immunization experiments [41 ]; they are locally confined in suitable neighboring niches. Nevertheless, the possibility that they arrive via the circulation in a manner similar to that demonstrated for LP PC [7 ] cannot be ruled out. Therefore, tonsil resident PCCOLL could be a temporal reservoir of successive locally induced humoral responses that performs a defensive function against recent pathogens from the oral cavity. Finally, tonsil PCCOLL have reached a differentiation stage that is close (HLA-DRlow, Bcl-2+, CD95+/–), but not identical, to that of fully mature BM PC, as these latter cells are CD20–, CD19+/–, CD45low [16 ]. This observation raises the possibility that PC niches occurring in different organs may differ in their PC supportive capacities. Further understanding of the PC biology will probably require a more detailed knowledge of the cellular and molecular components of the different PC niches.


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ACKNOWLEDGEMENTS
 
This work was supported by grants PI052357 and PI052406 from Fondo de Investigaciones Sanitarias of Spain.

Received May 4, 2007; revised July 12, 2007; accepted July 16, 2007.


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