Eur. J. Immunol. 1990.20: 1767-1773 Ontogenic analysis of thymic B reUs 1767
Jose L. Andreu-Sanchez·b.O,Jose FaroD,Jose M. Alonso·,Christopher J. Paige"',Carlos Martinez-A.·· andMiguel A. R. Marcos·'"
Centro de Biologia Molecular·,Universidad Autonoma, Servicio deReumatologiab., Clinica Puerta deHierro, Madrid, United'lmmunobiologieD, InstitutPasteur, Paris and the OntarioCancer Institute"', Toronto
Ontogenic characterization of thymic BIymphocytes. Analysis in different mouse strains*
We have characterized a population of murine B Iymphocytes present in thethymus (TBL). They are a minor subset (0.2%-1 % of total thymocytes), presentfrom perinatal periods onwards and constituted by activated cells with a highproportion of Ig-secreting cells. They represent the first B Iymphocytes detectedthat secrete IgO after birth. Functional analysis reveals that the frequency oflipopolysaccharide-responding cells in TBL is 5- to lO-fold lower than in thespleen. TBL from adult mice did not show any significant difference in their VHrepertoire expression when compared to peripheral B Iymphocytes. Furthermore, we have been able to isolate a subpopulation of B220+IgM-CD3thymocytes whose putative B cell precursor potential needs to be directlyanalyzed. These and other findings support the intra thymic resident characteristics of TBL and suggest new ways of elucidating its physiological role in thecomplex selective processes occurring inside the thymus.
1 Introduction
After homing to the thymus, early T cell precursorsrearrange and express a TeR. Thereafter, they undergo aseries of events, mostly associated to positive and negativeselection based on MHC recognition and deletion of Tcellsbearing TcR products of certain Vl\ genes with highreactivity to self-encoded determinants. Multiple interactions, most probably TcR mediated, with various accessorycell types (nurse cells, BM-derived M<I>and dendritic cells)have been implicated in the successive steps of T cellselection. The significant contribution of minor cell subsetshas been stressed, especially because of the finely designedthymic architecture and the highly variable rates of cellturnover inside the thymus [1].
Recently, we and others detected a population of functionalB Iymphocytes in normal thymuses, displaying characteristics which distinguished them from those present in peripheral organs. In fact, at least a fraction of them is locatedinside the thymic parenchyma, clearly questioning its roleas a recirculating subset [2-6]. Furthermore, B cells havebeen shown to play a significant role in influencing matureT cell repertoires [7-12]. Takingiiito account the intrathymic nature of most Tcell selection events, it seemed to us thatthe biological significance of TBL required a comprehensive experimental approach. Here we report a comparativestudy ofTBL in immunologically distinctive mouse strains.An immunodeficient (CBAIN) and two lupus-prone(MRL+/+ and MRLlpr/lpr) strains were analyzed. MRLmice develop a genetically determined autoimmune diseaseexpressed as a generalized B cell hyperactivity leading tohypergammaglobulinemia, production of autoantibodies,
[18186]
* This work was supported by grants from FlSS, NATO andCICyT.
• Supported by a fellowship from Fundacion Juan March.o Supported by INSALUD-Madrid.
Corr~spondence: Carlos Martfnez-A., Centro de Biologfa Molecular, CSIC, Universidad Aut6noma, Campus de Cantoblanco,E-28049 Madrid, Spain
formation of immunocomplexes and a variety of histopathologic manifestations of which glomerulonephritis isthe most prominent [13]. MRLlprllpr mice are characterized by an earlier onset of autoimmune disease (3- to6-month old vs. 2-year old) and a massive LN enlargement(Ipr phenotype) produced by the accumulation of doublenegative (CD3+CD4-CD8-) T cells. CBAIN mice areknown to suffer an X-linked immunodeficiency (xid)characterized by low serum IgM and Ig03 levels, theirinability to respond to certain TI antigens and low responsesto TO antigens [14].The fact that both the autoimmunity ofMRLlpr/lpr mice and the immunodeficiency of CBAINmice are thymus dependent as an added reason to use themin the analysis of an intrathymic population of B lymphocytes. The present studies, in both neonatal and adult mice,reveal features formally distinguishing thymic B lymphocytes (TBL) from splenic B cells.This report also providesevidence that TBL is a resident intrathymic populationhoming to the thymus early in ontogeny, and probablyundergoing in situ part of its B cell differentiation process.
2 Materials and methods
2.1 Animals
BALB/c, C57BL/6, MRL +/+, MRLlprllpr and CBAINxid deficient mice and Lewis rats were purchased from TheJackson Laboratories (Bar Harbor, ME) and maintained inour animal breeding facilities.When purification steps wererequired, thymocytes from 20-30 5-day-old newborn and 8to 12-week-old adult mice per experiment were pooled.Four-week-old Lewis rats were used as source of thymocytes (feeder cells) for LD assays.
2.2 Cell suspensions and PFC assay
Mice were killed by CO2 inhalation. Thymic lobes werecarefully removed from adjacent LN, avoiding bloodcontamination. Cell suspensions of thymocytes and splenocytes were obtained following standard procedures. Theprotein-A PFC assay has also been previously described[15].
00 14-2980/90/0808-1767$3.50 + .25/0
1768 J. L. Andreu-Sanchez, J. Faro, J. M. Alonso et al.
2.3 Purification of B Iymphocytes
Cell suspensions were immunoselected twice by incubationwith 111 mAb ([16]; anti-Thy-l) for 40 min at 4°C followedby treatment with rabbit complement (Behring-Werke,MarburglLahn, FRG for 30 min at 37°C). After removal ofcell debris with a Pasteur pipette, the remaining cells werewashed and resuspended in BSS-3% FCS. Depletionexperiments were routinely controlled by FCM analysis.
2.4 Proliferation, LD and ELlSA-spot assays
Cell cultures were done as previously described [17].Briefly, triplicate cultures of 1()5 cells/well were incubated inRPM! 1640 medium (Gibco, Grand Island, NY) supplemented with a selected batch of FCS at 10%, 50 ~M 2-ME,10 mM Hepes and antibiotics. LPS from E. coli 055: 85(50 ~g/ml; Difco, Detroit, MI) and Con A (5 ~g/ml; Pharmacia, Uppsala, Sweden) were used as Band T cellmitogens, respectively. Cell proliferation was measured ondays 2 to 5 of culture by dThd uptake after a pulse of1 ~Ci = 37 kBq [methyJ-3H]dThd (Radiochemical Centre,Amersham Int., Amersham, GB) during the last 12 h ofculture. Results are expressed as the mean of triplicatecultures, the SEM being < 10% of mean values.'.
Limited numbers of splenic B cells and Thy-1- thymocytesfrom 8-week-old BALB/c mice were distributed in 0.2-mlcultures containing growth-supporting rat thymic cells incomplete medium and 25 ~g/ml of LPS. For each frequencydetermination, 10 different cell concentrations were set upwith 48 replicates per point. Negative standards were a setof 48 cultures receiving only rat thymic feeder cells and LPS.The clonal size of Ig-secreting cells was calculated by usingthe data from cultures that, according to the theoreticanalysis, contained one responding cell/well. The formulac = (L PFC)IN was applied, where c is the average clonesize; L PFC is the total number of E-PFC found in thepositive wells and N is the theoretic number of clones inthose wells, which is obtained from the frequency ofprecursors in them.
The ELISA-spot assay was performed as previously described [18]. Polystyrene flat-bottom plates were coatedwith purified sheep anti-mouse Ig antiserum (5 ~g/ml) byovernight incubation at 4°C. LD lymphocyte cultures werefurther added to the plates which were then incubated for3-5 h (3rC, 5% CO2), The plates were subsequentlyincubated with purified biotin-labeled sheep anti-mouse Igantiserum in PBS-BSA (1% )-Tween (overnight, 4°C),avidin-labeled alkaline phosphatase (Sigma, St. Louis,MO) in PBS-BSA-Theen (1 h, 3rC) and the substrateagarose mixture. Between every step of the assay, the plateswere vigorously washed with PBS-Tween. The blue spotsgenerated after the last incubation were counted.
anti-V~6 (clone 44.22.1; [21]), and anti-B220 (cloneRA3.3A1; [22]) mAb were used as biotinylated reagents.followed by PE-streptavidin (Becton Dickinson). Beforestaining and to block nonspecific binding to FcR, cellsamples were incubated with 10% normal mouse serum(20 min, 4°C) in PBS-1% FCS-O.l% sodium azide. Afterwards, they were incubated with the biot.inylated and/orFITC-conjugated mAb (20 min, 4°C), followed by PEstreptavidin, when required. The relative fluorescenceintensity of individual cells was measured using the EpicsProfile Flow Cytometer (Coulter Electronics, Hialeah.FL), after gating of viable lymphoid cells by forward lightscatter vs. propidium iodide staining. Background valueswere defined by isotype-matched irrelevant Ab substitutingthe specific ones. Data were collected from lot cells/samplewith a logarithmic fluorescence scale. The two-color graphicdisplay was generated by F1owSys, software developed byDr. L. Pezzi at our institution.
2.6 B cell colony assay
Spleen cells and Thy-1- thymocytes were cultured indouble-layer colony, as described previously [23]. Thebottom layer containing 1 ml of OPTI-MEM medium(Gibco), supplemented with 7% FCS, 0.5% melted BactoAgar (Difco), 50 ~g LPS and 1% washed SRBC. Spleencells or Thy-1- thymocytes were resuspended in the samemedium lacking LPS and SRBC, and plated as top layer.The cultures were incubated at 37°C in humid air contain
ing 5% CO2 for 5-6 days.
The colonies growing in the agar disc were transferred tonitrocellulose filter GenScreen Plus (New EnglandNuclear, Boston, MA) as described [24]. Thereafter thefilters were prehybridized for 12-24 h at 42°C in a mixturecontaining 50% formamide, 5 x SSPE and 4 x Denhardt'ssolution. After hybridization (the same mixture as beforecontaining 1 x 1()6 cpm/ml of [32P]CTP-labeled probe) for48 h at 42°C, the filters were washed, dried and autoradiographed with intensifying screens (Ilford, Mobberley, GB)at -70°C for 1-2 weeks. The dots of colony-forming unit(CFU)-B were enumerated, and the relative representationof each gene family was plotted as the percentage of totalVH gene expression. Plasmids and fragment DNA wereobtained and used as previously described [24, 25].
3 Results
3.1 Quantitation of background PFC in the thymus:analysis throughout development
We have previously reported the presence of considerablenumbers of spontaneous Ab-secreting cells in the thymus ofnormal and germ-free mice [2]. When compared to splenocytes, TBL showed a clear predominance of IgG and IgAwith respect to IgM isotypes. Here we confirm and extendthose results by an ontogenic and comparative analysis ofimmunologically distinct mice (Table 1). Thus, whereas theIgG/lgA bias was evident for BALB/c and xid-deficientCBAIN adult TBL, this was not apparent in the case ofautoimmune MRL mice. In the latter, the PFC isotypepattern of both TBL and splenic B cells was ,rather similar.
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EUT.J. Immunol. 1990.20: 1767-1773 Ontogenic analysis of thymic B cells 1769
While PFC assays revealed the number of Ab-secretingcells, a more detailed characterization of B Iymphocytesrequired the identification of the markers expressed.However, the low number of B cells actually present in thethymus « I % in normal mice) caused a new problemrequiring some purification. In order to enrich these cells,two successive steps of negative selection of Thy-l +thymocytes were performed. The proportion of recoveredcells after killing was consistently about 0.2%-1 % of totalthymocytes. FCM analysis revealed that the recoveredpopulation, while showing a depletion of Thy-I + T cells inthe spleen « 1%), always revealed a remaining subset ofThy_ldulJ thymocytes (5%-15%; Fig. 1). A better characterization of this recovered thymocyte population showedthat, in addition to the expected subsets ofIgM+ and non-T,
3.2 Quantitative FCM analysis of mouse TBLmost likely reflecting the intensive and abnormal B cellstimulation in the periphery of these mice.
""urthermore, when early ontogenical stages (5 days post"irth) were analyzed, clear-cut differences were observedbetween TBL and splenic B Iymphocytes (Table I). Themost consistent finding was the secretion of Ig isotypesIgG2b, IgG3 and IgA by the TBL, which were still notproduced by the spleens of the same mice. This defines astrict distinction between B cell populations present in thetwo compartments, at least at that stage. Thus, with theexclusion of the newborn MRLlprllpr mice, which alwaysbehave in a more diverse way with respect to isotypeexpression, the rest of the strains analyzed always showedan earlier class switching to IgG by TBL than by splenic BIymphocytes. Whether this is an intrinsic characteristic ofthe B cells or is dependent on cell regulatory influences is anopen question that present in vitro experiments are tryingto elucidate. At the neonatal stage the CBAIN strainspontaneously secretes only IgM in the spleen whileshowing a pattern of Ig production in the thymus incommon with the other analyzed mouse strains. It is likelythat, as described in Peyer's patches [26], B cells from xidmice are pushed in the thymic environment to further~tages of differentiation under specific T cell influences.
'.
These data support the view that: (a) TBL are present inthe thymus from very early in life; (b) they probably are thefirst B lymphocytes in mouse to undergo IgG class switching; and (c) given the Tcell-dependent nature of some of theisotypes produced, they need to be locally induced to thisproduction by intrathymic immunocompetent T cells.
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When the TBL were derived from adult thymuses, theisotype pattern of Ig-secreting B cells was rather similar tothe one produced by splenic B cells, except for the knownrelative increases in IgG and IgA Ab, found here inBALB/c and CBAIN mice (Table 1).
Figure I. FCM characterization of BALB/c Thy-l:- thymocytesafter negative selection with anti-Thy-l mAb and complement. Thenumber in the top corner indicates the proportion of positive cellsin each population when background fluorescence (broken line) issubtracted. The staining was undertaken as described in Sect. 2,and dead cells were gated out with propidium iodide.
Table 1. Ontogenic analysis of spontaneous PFC in mouse thymus and spleen
a) The data shown are the mean of three individual experiments with duplicated tests in each isotype, the SEM being < 10%.b) Pools of 5-day-old mouse thymocytes.c) Two- to three-month-old mice.
1770 J. L. Andreu-Sanchez, J. Faro, J. M. AJonso et al.
non-B cells, the immunoselection procedure had selectedthe minor component of CD3+TcRa/~+CD4-CD8- thymocytes, expressing skewed repertoire towards the use ofV~8 gene products. The data from a representative experiment with BALB/cThy-l- thymocytes are shown in Fig. 1and were confirmed in two-color analyses (data not shown).A variable percentage of IgM+ cells was always foundamong the Thy-l- thymocytes of the analyzed strains(Table 2). If one assumes the same nonspecific cell losses asa consequence of the purification steps for both neonataland adult cell suspensions, the percentages obtained in theFCM studies can be referred to the original thymocytepopulation after considering the absolute cell recovery ofimmunoselection procedures; these numbers are shown inTable 2.With the exception of the autoimmune MRL lprllprmice, we could not detect significant increases in thenumbers of thymic IgM+ Iymphocytes from newborn toyoung adult mice. These findings argue against a progressive accumulation of recirculating B cells in the thymus.
'.
Figure 2. Two-eolor FCM analysis for IgM and B220 expression ofThy-I-depleted thymocytes. Graphic display was made usingFlowSys program.
Table 2. FCM quantitation· of IgM+ lymphocytes in the thymus
Eur. J. Immunol. 1990.20: 1767-1773
When the ratio of number of Ig-secreting cellslIgM+ cells isanalyzed in TBL of different mouse strains during differentlife periods (Table 2) and compared to that in spleen BIymphocytes (data not shown), a major difference isevident. Thus, in adult BALBIc this ratio is 1/6 for TBL and1/111 for splenic Iymphocytes. It appears that a largefraction of TBL is in terminal stages of differentiationbecause of the constitutive Ig secretion. Also, a clearincrease in the ratio of Ig-secreting cellslIgM+ is evidentfrom newborn to adults (5-20 times greater; Table 2).Subsequently, and despite their low numbers, TBL probably contribute in a substantial way to the pool of Ig in theserum. In agreement with this, many of the TBL areincluded in the CDS+ B cell population ([5] and ourunpublished results), which has been shown to be responsible for the basal levels of natural Ab [27].
Two-color FCM analysis of B220 vs. IgM revealed a smallbut defined subpopulation of B220+IgM- cells among theThy-l- thymocytes (Fig. 2). This subset does not expressany Tcell marker (Thy-I, CD3, CD4, COS; data not shown)and is phenotypically suggestive of a pre-B lymphocyte[22]. Within the sensitivity limits of FCM, this subset doesnm express sIgM and, in fact, it was separated fromB220+IgM+ cells by at least 120 channels of fluorescenceintensity. The possibility that these cells have a very lowdensity of sIg, as has been suggested for recent BM B cells[28], cannot be ignored. Ongoing studies of in vivo celltransfer and in vitro culture will clarify the B cell progenitorpotential of these thymocytes. The existence of TBLimmediately after birth suggested that (if the consecutivewaves of embryonic thymic re population in the mouse [29]
. also account for precursors of this population) they wouldalready home to the organ in the first wave of lymphoidprecursors (days 10-14 of fetal development). Thereafter,they could perform at least part of their commitment/differentiation program in situ.
3.3 In vitro proliferation of TBL
We have performed functional analyses of TBL by mitogenic stimulation (Con A, LPS; Fig. 3). Consistently, TBLshowed a limited LPS-dependent proliferative responsewhich was ten times lower than that of splenic B lymphocytes. The Thy-l- thymocyte cultures responded to Con Astimulation, probably due to the presence of the doublenegative population in the cell suspensions.
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a) As defined in Table I.b) The proportion of IgM+ cells was calculated by subtrac
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c) The numberofIgM+ cells was calculated by the formula:[% IgM+ cells of the Thy-l- pool obtained from 1()6
thymocytes) x [number of recovered cells after ThyI-negative selection)Il00.
Eur. J. Immunol. 1990.20: 1767-1773 Ontogenic analysis of thymic B cells 1771
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Figure 3. Kinetics of the in vitro proliferative response of TBL vs.splenic B Iymphocytes: LPS (6); Con A (0); medium (0). (A)Pool of Thy-1- thymocytes in which B cells represent 40% of thetotal population; (B) total splenic cells; (C) splenic B cells (> 80%sIg cells). All results are the mean of triplicated cultures with SE<10% (not shown).
As shown above, TBL display a number of unique characteristics such as early IgG isotype secretion and C05expression. The possibility of differential VH repertoireexpression by this cell population was analyzed by an RNAcolony blotting'assay which detects the RNA from as few as20-30 cells. As shown in Fig. 5, there is no indication for adifferential V H usage between B cells derived both fromspleen and thymus in the two strains analyzed. As thesestudies were done in adult mice, the rare possibility ofparticular differences in newborns still remains (studies inprogress). Also, the frequency of VH family usage appearsto be a genetic trait showing strain-dependent polymorphism.
4 Discussion
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The present study has evidenced several distinctive featuresof TBL vs. splenic B lymphocytes which argue against theclassical interpretation of TBL as a purely recirculatingsubset, but support their consideration as a resident thymicsubpopulation. Recently, based on results using differentexperimental systems, others have also supported this idea,and advanced putative roles for these B cells in thymicTcellselection. TBL are a functional population from very soonafter birth (we have not analyzed the embryos). At thisstage, they seem to be the first B lymphocytes that secreteIgG, which suggests either a different B cell subset or, more
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The frequencies of LPS-responding B cells in both splenicand thymic B cell populations from BALBIc mice werequantitated in parallel in LD analysis (Fig. 4). While thefrequency of LPS-responding B cells was 1 : 7 (1 : 6-1: 9) inspleen, it was only 1: 190 (1: 155-1 : 243) among TBL.These data explain the low proliferative responses previously found in bulk cultures. However, the average clonalsize, 9-11 cells/clone, was the same for both splenic B cellsand TBL. These results demonstrate that the lower proliferative response to LPS of TBL vs. splenic B cells in bulkcultures is due to a lower frequency of LPS-respondingB cells among TBL.
Figure 4. LD assays for frequencies of clonal precursor B cells inTBL (e) and splenic B cells (0), analyzed by ELISA-spotassays.
3.4 V H gene family expression in TBL vs. splenicB lymphocy1es
Previous studies have demonstrated that CFU-B coloniesare derived from single IgM-bearing cells and that onefourth of splenic B cells are able to form a detectablecolony. Therefore, the analyzed cells seem to be representative of the total B cell population samples [23].We havecharacterized the abilityofTBL to form CFU-B, both at theaqult and newborn stages in normal mice. In agreementwith the data of Ab-secreting cells and IgM+ cells, ontogenic characterization of CFU-B both in newborn (2-d ayold) and adult (8- to IO-week old) show a similar number ofTBL during dev~lopment (data not shown).
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Figure 5. VH family usage of LPS-stimulated Thy-l - thymocytes(.) and splenic cells (Ilill)from adult BALB/c-(A) or C57BL/6 (B)mice. Each bar shows the frequency of each VH family as apercentage of the total VH gene expression. Results shownrepresent the mean of three different experiments.
1772 J. L. Andreu-Sanchez, J. Faro, J. M, Alonso et al. Eur. J. Immunol. 1990.20: 1767-1773
probably, a specific "helper" microenvironment, in whichintrathymic immunocompetent Tcells play a critical role. Infact, throughout the whole life-span, a large fraction ofTBL is pushed into terminal stages of differentiation (ten toone hundred times the splenic B Iymphocytes), a very largeproportion of them producingTcell-dependent Ig isotypes.Present data differ substantially from those reported byInaba et al. [5], probably due to the low sensitivity of thedeveloping antisera used by these authors since, not only inthe case of thymic B cells but also when splenic B cells weretested for PFC, their numbers were rather low whencompared with those in our study and in previous works [2].We have also characterized the low responsiveness in vitroof TBL to LPS, which is due to a low frequency ofLPS-responding B cell precursors as LD analysis shows andnot to putative cell suppressive influences in bulk culture as .recently suggested [5]. The selective predominance of theCD5+ B cell population in the mouse thymus has also beenreported [5], as well as the very high percentage ofproliferating BrdUrd+ cells among rat TBL [30] whencompared to other cell subsets and organs. These datasuggest a very active role of this subset in intra thymiccellular events. In terms of VH gene family expression, wecould not detect major differences between the repertoireof adult TBL and splenic B cells. Altogether, these findingsdefine a specific thymic B cell population which is not amere representation of the predominant B cells in theperiphery. Furthermore, we have observed a minor subpopulation of B220+IgM-Thy-l- thymocytes, phenotypicallysuggestive of B cell precursors, an aspect which is beingdirectly addressed in both in vivo and in vitro systems.Interestingly, systems of in vitro viral transformation ofimmature lymphoid cells have already detected B cellprecursors in fetal thymus [31].
Related to the physiological role of TBL, we envisage twowell-developed lines of research. On the one hand, thedeletion of Vjl TcR gene products upon interactions in thethymus with appropriate antigenic specificities has beendemonstrated [32,33]. Whereas the requirement of Agexpression by corticoepithelial cells for positive selection ofdifferentiating TcR + thymocytes has been shown, no intrathymic APC has been definitively involved in the clonaldeletion process. In a majority of experimental modelswhere this deletion has been shown, there are two commonfeatures: (a) the selective process of deletion occursintrathymically [34] and (b) B Iyniphocytes seem to berequired in the process. Thus, specific reactivity to Mlsantigen is only evidenced against stimulator B cells [35, 36],and although V1\17aTcR recognize I-Ek expressed by both BIymphocytes and other accessory cells, V1\17aT cell hybridomas respond very poorly to thymic cortical epithelialcells [37], and these specificities were eliminated only whenconfronted with I-Ek in the presence ofB lymphocytes [38].Clearly, TBL represent the best candidate to be involved inthe described phenomena. In fact, TBL are located at thecortico-medullary jUl1ction, which defines the unselected/selected populations of TcR in the thymic tissue sections, and form typical B cell structures (germinal centers)[2-6]. Recently, we have further purified sorted by FCMboth IgM+ TBL and other intrathymic IgM- APC popuJations. Both populations provided a good stimulus inH-2-incompatible MRL when used as stimulators in in vitroMRL cultures. However, only TBL were able to induce agood proliferation of LN cells in Mls-incompatible condi-
tions (DBA/2 TBL to BALB/c LN cells; J. Faro et aI.,submitted). In the light of these data, the possible role ofminor subsets of I-Ek' B Iymphocytes in the described I-Etransgenic mice <:annot be disregarded [39].
On the other hand, it has been shown that positive selectionof potential T cell repertoires is influenced not only byMHC antigens but also by Ig and/or B cell repertoires. Inparticular, neonatal and naturally activated B lymphocytes,including CD5+ cells, are relevant in this respect [40, 41].Previously considered as peripheral to T cell selection, itnow appears that TBL can also pal ticipate in this selection.There is substantial evidence supporting the mutual cellinteraction between neonatal-like CD5+ lymphocytes andfunctional T cell precursors in order to obtain a furtherrepertoire diversification [42]. Obviously, if many of thedescribed T cell selection events are dependent upon therecognition of specific pep tides [32], TBL still constitute arelevant population to be considered: (a) because theirhighly activated state probably provides an excellent machinery for antigen processing and presentation; and (b)because of the specific ability of B lymphocytes to presentself-Ig in the form of idiopeptides [43]. The selectiveTBL-dependent proliferation of self-idiotype (Sp6)-specific thymocytes has recently been characterized in in vitrrstudies [44].
Some final points should be stressed to establish therelationship between emergence of autoimmune diseasesand the selective expansion of thymic B lymphocytes, bothfrom experimental and clinical perspectives [13, 45]. Insome cases, this occurs in the context of a real thymichypertrophy, like in myasthenia gravis, which is directlyinvolved in the clinical evolution since adult thymectomydramatically alters the myasthenic symptoms [46].Although there are some insights related to the Ig dependence of these phenomena [47], the complex cause-effectrelationships involved and their relevance to autoimmunepathogenesis remain open and exciting questions. Presently, we cannot elude the formal paradox created by theexpansion in autoimmune diseases of a thymic populationwhich is most likely implicated in the clonal deletion ofautoreactive T cells and tolerance.
We would like to tJw.nk E. Leonardo and J. M. Gonzalo fortechnical assistance and M. Messman for help in the preparation cthe manuscript.
Received December 15, 1989; in revised form March 28, 1990.
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