the murine autologous mixed lymphocyte response: distribution of stimulator cells

Journal of Autoimmunity (1995) 8, 21–31

The Murine Autologous Mixed LymphocyteResponse: Distribution of Stimulator Cells

James E. Riggs, Gary R. Sirken, Lisa G. Prior andMonte V. Hobbs*

Department of Biology, Rider College, Lawrenceville, NJ 08648-3099 and*Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037,

USA

(Received 15 April 1994 and accepted 21 September 1994)

Previous studies have shown that neonatal, but not adult, murine thymic Tcells proliferate when co-cultured with syngeneic, adult splenic B cells.Evidence suggests that expansion of such self-reactive T cells precedestheir clonal deletion or functional inactivation (anergy). This mechanismmay be of particular significance for establishing peripheral (extrathymic)tolerance. In order to assess the autostimulatory capacity of B cells presentin a variety of lymphoid tissues, neonatal T cells were cultured with Peyer’spatch and peritoneal cavity cells. The results indicate that B cells in thesetissues readily induce T cell proliferation. Evidence suggests that the B-1 Bcell subpopulation is not obligatory for this process. Self-reactive T cellswere evident in the spleen of young mice, substantiating a role for B cells inmaintaining peripheral tolerance. The results suggest that B cells involvedin this process are distributed in a variety of lymphoid organs.

Introduction

Deletion and functional inactivation of self-reactive T lymphocytes are requisite toavert autoimmunity. Apparently, these processes are less efficient early in develop-ment as self-reactive T cells are found in the thymi of mice shortly after birth [1–4].Organ-specific autoimmunity in neonatally thymectomized mice indicates thatautoreactive T cells are not restricted to the thymus (THY) [5, 6]. Peripheral(extrathymic) tolerance is essential for controlling these autoreactive escapees ofclonal deletion [2]. The cells that induce peripheral tolerance, and their anatomicaldistribution, comprise a major branch of tolerance research. B lymphocytes are

Correspondence should be addressed to: J. E. Riggs, Department of Biology, Rider College,2083 Lawrenceville Road, Lawrenceville, NJ 08648-3099, USA.

210896-8411/95/010021+11 $08.00/0 ? 1995 Academic Press Limited

efficient tolerogenic antigen-presenting cells (APCs), particularly for naive T cells[7–12]. In the murine autologous mixed lymphocyte response (AMLR), however,neonatal T cells proliferate when cultured with syngeneic, adult spleen (SP) B cells[3, 4, 13]. B-cell expansion of self-reactive T cells may reflect an interaction thatprecedes tolerance [14, 15]. Consistent with this notion, the age at whichstimulatory SP B cells become active in the murine AMLR correlates with the ageat which T cell responsiveness declines [16].Determining the tissue distribution and particular subpopulations of stimulatory

B cells could reveal sites of, and cells associated with, peripheral tolerance. Alimited analysis of the tissue distribution of B cells stimulatory in the murine AMLRhas been conducted [16, 17]. Lymphocytes from Peyer’s patches (PP) and theperitoneal cavity (PerC), putative sites of extrathymic T cell differentiation [18,19], and the autoreactive B-1 [20] or J11d" ‘memory’ [21] B cell subpopulations,have not been tested [4]. In this report, the AMLR-stimulatory capacity of these Bcell sources and B cell subpopulations were tested. AMLR-stimulatory B cells werefound equally distributed in SP, PerC, and PP, negating a specific role for the B-1B cell subpopulation in the AMLR. These findings are discussed in the context ofB and T lymphocyte interaction during development.

Materials and methods

Mice

BALB/c and BALB.xid (X-chromosome-linked immunodefective) mice (Igha,H-2d, Mlsb), C.B-17 and C.B-17 scid/scid (severe-combined immunodefective)mice (Ighb, H-2d, Mlsb), DBA/2J mice (Ighe, H-2d, Mlsa), and C57BL/6J andC57BL/6J. mev (motheaten viable) mice (Ighb, H-2d, Mlsb), bred and maintained atRider College, were used between 3 days and 32 weeks of age. MEV mice werederived from breeding pairs obtained from the Jackson Laboratory, Bar Harbor,ME. All other strains were derived from breeding pairs provided by Raquel Davisand Donald Mosier, The Scripps Research Institute, La Jolla, CA. All mice werehoused and handled in accord with NIH guidelines.

Cell preparations

SP, PP, and THY cell suspensions were prepared by mincing between sterile glassslides in HBSS. RBCs were removed from SP cell suspensions by osmotic shockwith Tris-buffered ammonium chloride [22]. PerC cells were collected by flushingthe peritoneum with 10 ml of warm (37)C) HBSS. SP, PP, and PerC cellsuspensions were T cell-depleted using HO-13-4 MAb (rat IgM anti-mouse Thy1.2 [23]) plus baby rabbit complement (Pel-Freeze Biologicals, Rogers, AK)treatment followed by density gradient centrifugation (Lympholyte M, AccurateChemical, NY) to retrieve viable cells. As total THY cell preparations enriched foradult T cells afford better MLRs [24], CD4+8" thymocytes were enriched bydepleting CD4+8+ and CD4"8+ T cells by two rounds of 3.155 MAb (rat IgManti-mouse CD8 [25]) plus complement (Low Tox M, Accurate Chemical)treatment followed by density gradient centrifugation. RBC-depleted SP, PP, and

22 J. E. Riggs et al.

PerC cells were depleted of J11d+ cells by two rounds of J11d MAb (rat IgManti-mouse J11d [26]) plus baby rabbit complement treatment. To prepareperipheral CD4+ T cells, RBC-depleted SP cells were depleted of B and CD8+ Tcells by two rounds of treatment with J11d and 3.155 MAbs plus baby rabbitcomplement treatment followed by density gradient centrifugation. The latterprocedure routinely resulted in recovery of 27.5&1.5 (SE)% of input SP cells from7-week-old mice and 5.0&1.9% perinatal SP cells from 2-week-old mice.

AMLR cultures

Responder T cells were suspended in RPMI 1640 culture medium (Life Technolo-gies Inc., Grand Island, NY) supplemented with 10% heat-inactivated FCS(Hyclone, Logan, UT), 1 ìg/ml gentamycin, 100 u/ml penicillin, 100 ìg/ml strep-tomycin, 5#10"5 2-mercaptoethanol, and 2 m L-glutamine. Responder Tcells (2#105/well) were cultured with 4.0, 2.0, 1.0, 0.5, or 0.25#105 SP, PP, orPerC B cell-enriched stimulator cells in 96-well culture plates (3696 A/2; Costar,Cambridge, MA). Plates were incubated in a humidified atmosphere of 5% CO2 at37)C for 72 h. One ìCi/well of [3H]thymidine was then added and wells wereharvested 17 h later with a cell harvester (Skatron Inst., Sterling, VA). Radioactivitywas measured by scintillation spectrometry and results are expressed as counts perminute (cpm) &SEM for n=4 wells/sample tested. All T cell preparations werecultured with Mls-disparate DBA/2J stimulator cells as proliferation controls. Asnoted in the original description of the AMLR [4], experiments using mitomycin-Ctreated stimulator cells (SP, PP, or PerC) confirmed that T cells constitute thethymidine-incorporating subpopulation in all systems described herein.

Serum IgM ELISA

The procedure followed was identical to that described previously [27].

Results

PerC and PP cells stimulate neonatal T cell proliferation

The murine AMLR is characterized by proliferation of neonatal, but not adult,Class II MHC-restricted T cells after co-culture with syngeneic, adult SP B cells [3,13, 16, 28]. Prior surveys of the tissue distribution of stimulatory B cells found thatSP cells were more effective than lymph node, bone marrow, and THY cells forinducing T cell proliferation [13, 16]. B cell sources and distinct B cell sub-populations not studied in the murine AMLR include PerC cells, enriched for B-1B cells, and PP cells, which lack the B-1 B cell subpopulation [4, 20, 29, 30]. Theability of these B cells sources to generate the AMLR were compared with that ofSP cells. Both PP and PerC cells were stimulatory; however, PerC cells inducedlower levels of neonatal T cell proliferation and were inhibitory when plated at ahigher density (2#105 cells/well) (Figure 1). That adult T cells did not proliferatewhen co-cultured with any of the stimulator cell preparations is consistent withprior descriptions of the AMLR [3, 4, 13, 17]. Adult T cells are fully functional,

Distribution of AMLR-stimulatory B cells 23

however, as they proliferate when cultured with Mls-disparate (DBA/2J) spleencells (not shown). These results reveal that AMLR-stimulatory cells are distributedin a variety of peripheral lymphoid tissues and anatomic locations.

B cells are responsible for the T cell proliferation induced by PerC and PP cells

As prior studies have shown that B lymphocytes are the cell type required forgenerating the murine AMLR [13], they are likely to be the stimulatory populationin PerC or PP preparations. To test this, PerC, SP, and PP cells from Bcell-defective XID [31] and B and T cell-defective SCID [32] mice were employedas stimulator cells in the AMLR. As shown previously with SP cells [33], PP andPerC cells from XID mice fail to induce significant levels of neonatal T cellproliferation (Figure 2). SP and PerC cells from lymphocyte-deficient SCID micealso fail to generate the AMLR (PP are absent from SCID mice and thus were nottested). However, SP and PerC cells from XID and SCID mice reconstituted withnormal (C.B-17 or BALB/c) B cells generate the AMLR (Figure 3). These resultsillustrate that B cells are the AMLR-stimulatory cell in all tissues tested herein.

Self-reactive T cells are found in the periphery of young mice

A caveat of the AMLR is that the responding cells, mature CD4+8" lymphocytes,have never been shown to exit the thymus. To determine whether self-B cell-reactive T cells are evident in the periphery of young mice, splenic CD4+8" T cells

Figure 1. Neonatal (/) and adult (.) T cell proliferation induced by SP, PP, and PerC cells fromnormal mice. Stimulator SP, PP, and PerC cells were cultured, at the numbers listed, with 2#105

4-10-day-old (neonatal) and 42–56-day-old (adult) BALB.xid CD4+8" thymocytes. Results arepresented as the average CPM of a minimum of five cultures (n=4 wells/culture).


were tested as responder cells in the AMLR. XID mice were used as the source ofperipheral T cells because their B cell maturation defect [34] facilitates purificationof splenic CD4+8" T cells [35]. Responses to the Mlsa superantigen found onDBA/2J SP cells were included in these experiments as a control for T cell function.

Figure 2. Neonatal T cell proliferation induced by SP, PP, and PerC cells from XID (.), SCID (.),and normal mice (/). Stimulator cells were cultured at 2.0 (SP and PP) or 1.0#105 (PerC)/well with2#105 4–10-day-old BALB.xid CD4+8" thymocytes. Results represent the average cpm of a minimumof three cultures (n=4 wells/culture).

Figure 3. Neonatal T cell proliferation induced by SP and PerC cells obtained from B cell-reconstituted (/) XID (a) or SCID (b) mice. 2.0#105 SP and 1.0#105 PerC cells from XID or SCIDmice, injected i.v. 8 weeks previously with 107 C.B-17 or BALB/c SP cells, were co-cultured with2#105 4–8-day-old BALB.xid CD4+8" thymocytes. Results represent the average cpm of a minimumof three cultures (n=4 wells/culture). Controls (.) represent age-matched, unreconstituted XID andSCID mice.


SP and PerC B cell-reactive T cells were evident in the thymi of 2-week-old miceand relatively absent from both spleens and thymi of 7-week-old mice (Figure 4).The spleens of 2-week-old mice, however, harbored PerC B cell-reactive T cells.Unlike the other groups tested, the proliferation evidenced by perinatal splenic Tcells was greater with PerC B cell stimulation than with Mls-disparate stimulation.These results are not unique to perinatal XID T cells as similar results wereobtained using normal mice as donors of splenic T cells (data not shown). Thisresult suggests that self-B cell reactive T cells may comprise a significant proportionof the perinatal T cell pool.

SP cells of motheaten viable mice fail to generate the AMLR

Due to their prominence in neonatal mice [29] and in the thymi of adult mice [36]and their specificity for autoantigens [30, 37], we speculated that B-1 B cells wouldbe an effective B cell subset for generating the AMLR. The lack of AMLRstimulation with B cells from XCID mice, which lack the B-1 subset [38],reinforced this hypothesis (Figure 2). PP cells from normal mice, however, lack theB-1 B cell subpopulation and generate the AMLR. B-1 cells alone, therefore, wouldappear to not be requisite for the AMLR. To define more rigorously the AMLR-stimulatory capacity of B-1 B cells, SP cells from MEV mice were cultured withsyngeneic, neonatal T cells. All B cells in mice homozygous for the mev gene have

Figure 4. Perinatal and adult thymic or splenic T cell proliferation induced by syngeneic (BALB/c) SP(.) and PerC (.), or Mls-disparate DBA/2J SP cells (/). 2.0#105 SP or 1.0#105 PerC cells werecultured with 2#105 CD4+8" THY or SP cells from 2 or 7-week-old BALB.xid mice (a) Thymus,7-week-old; (b) Spleen, 7-week-old; (c) Thymus, 2-week-old; (d) Spleen, 2-week-old). Results representthe average of four cultures (n=4 wells/culture). Excepting 2-week-old spleen, all T cell preparationsexhibited >100,000 cpm when cultured with DBA/2J SP cells.


been described as members of the B-1 subset [39]. Homozygotes exhibit charac-teristics ascribed to the B-1 subpopulation, e.g. high levels of spontaneous IgMproduction; heterozygotes have serum IgM levels similar to normal C57B L/6J mice(Figure 5(a)) [20, 39]. Compared with normal C57BL/6J and heterozygousC57BL/6J.mevv SP cells, homozygous C57BL/6J.mev SP cells are less effectivein generating the AMLR (Figure 5(b)). The low AMLR found in these cultureswas consistent with prior studies of the C57BL/6J and B-1-enriched NZBstrains [16, 40]. These data suggest that B-1 B cells are not required to stimulate Tcell proliferation.

J11d" PerC cells induce neonatal T cell proliferation

To investigate further the role of specific B cell subpopulations in the induction ofT cell proliferation, PerC, SP, and PP preparations were depleted of J11d+ cells.This marker was chosen as it is expressed on the majority of B cells [26]; J11d" Bcells are reported to represent the memory precursor and memory B cell pool [21].SP and PP preparations depleted of J11d+ cells had markedly reduced AMLR-stimulatory capacity whereas J11d" PerC cells remained stimulatory (Figure 6(b)).B cell depletion was confirmed by the abrogation of LPS responsiveness (Figure6(a)). These data reinforce the idea that B cells are the stimulatory subpopulationin the AMLR and reveal that J11d" PerC cells are stimulatory.

Discussion

By employing the murine AMLR to assess potential anatomic sites and cellsinvolved in the maintenance of tolerance, AMLR-stimulatory cells were found inthe SP, PerC and PP of normal, but not XID or SCID mice. B cells are thestimulatory cell type in these tissues as SP and PerC cells from B cell-reconstitutedXID and SCID mice generate the murine AMLR. An obligatory role for B-1 B cellswas negated by the failure of SP B cells from MEV mice, composed primarily of the

Figure 5. IgM levels (a) found in and neonatal T cell proliferation (b) induced by C57BL/6J andC57BL/6J.mev mice. Serum IgM levels of 6–12-week-old mice were measured by ELISA. 2.0#105 SPcells from each strain were cultured with an equal number of 3–5-day-old C57BL/6J CD4+8"

thymocytes. Results represent the average of three or more cultures (n=4 wells/culture).


B-1 subset, to generate the AMLR. Memory precursor cells and memory B cells(J11d") in the PerC, but not the SP or PP, were shown to be effective in theAMLR. That autoreactive T cells were found in the spleen of young mice supportsthe hypothesis that the AMLR represents an interaction that precedes the acqui-sition of extrathymic tolerance. Combined, the data suggest that distribution ofAMLR-stimulatory B cells throughout the immune system ensures the meanswhereby extrathymic tolerance may be established.That SP, PerC, and PP differ markedly in B-1 B cell composition [29] yet are

similar in AMLR-stimulatory capacity indicates that this B cell subpopulation is notrequired to generate the AMLR. This was surprising because characteristics of B-1B cells indicate that they could be potent in the AMLR: (1) prominence in thethymus [36], (2) absence from XID mice [38], and (3) specificity for autoantigens[30, 37]. Prior studies, however, had shown that NZB mice, noted for increasedrepresentation of the B-1 B cell subset [29], do not exhibit the AMLR [40]. Cellsfrom C57BL/6 mice exhibit a meager AMLR [16, 39] that we have shown is furtherconstrained by the mev mutation (Figure 5). Therefore, strain-specific variabilityand similar stimulatory capacity amongst SP, PerC and PP cells suggest that thepool of B cells that generates the AMLR does not have to include the B-1subpopulation. Rather, collectively, these observations suggest that B-1 B cells mayinhibit the murine AMLR. This hypothesis is currently being tested.Can a specific phenotype be ascribed to AMLR-stimulatory B cells? Prior studies

have shown that activated B cells are quite effective for the generation of allogeneicor Mls MLRs [41]. Activated B cells certainly are present in the SP, PP, and PerCof normal mice. In contrast, such cells are rare in lymph node and bone marrow,tissues relatively devoid of AMLR-stimulatory cells [16]. Defective B cell activationis a phenotypic hallmark of XID mice and thus, a potential reason for their lackingthe AMLR [31, 33, 42]. Furthermore, that xid B cells bind but fail to becomeactivated by polyvalent antigens [31] may provide insight as to the nature of theself-antigen(s) being recognized in the AMLR.

Figure 6. LPS response (a) found in and neonatal T cell proliferation induced (b) by untreated(control; .) and J11d-depleted SP, PP, and PerC cells (/). Control and J11d-depleted stimulator cellswere cultured at 2.0 (SP and PP) of 1.0#105 (PerC)/well with 25 ìg/ml LPS or 2#105 4–10-day-oldBALB.xid CD4+8" thymocytes. Results represent the average of three or more cultures (n=4wells/culture).


One possible consequence of B cell activation is memory cell generation. B cellsthat enter this differentiation state are long-lived and lose an epitope recognized bythe MAb J11d [26]. J11d" SP and PP cells failed to generate the AMLR whereasJ11d" PerC did, indicating that the latter may represent a distinct subpopulation.This anatomic restriction is curious yet consistent with studies showing thatperitoneal B cells are long-lived [29]. Prior studies have shown that maintenance ofimmunologic memory and tolerance requires the persistence of Ag and APC,respectively [43, 44]. Correlating these observations raises the possibility that adultT cells are unresponsive in the AMLR due to the presence of tolerogenic(stimulatory) B cells and that neonatal T cells are responsive due to their absence.This hypothesis is supported by prior studies indicating that the age at whichstimulatory B cells are first evident correlates with the age at which T cellresponsiveness declines [16, 35] and more recent work showing that B cellacquisition of the ability to induce IL-2 production occurs late during the perinatalperiod [45].In summary, we have shown that AMLR-stimulatory B cells, potentially respon-

sible for maintenance of extrathymic tolerance, are distributed in a wide variety oflymphoid tissues. These cells may play a significant role in maintaining self-tolerance in T cells that escape clonal deletion in the thymus. Abrogation of thisform of tolerance may precede the onset of autoimmunity.

Acknowledgements

We thank Daniel Spector for maintenance of our mouse colony and LauraBlinderman for critical review of this manuscript. This work was supported bygrants to J.E.R. from the NIH AREA program (R15 HD29206-01) and the NewJersey Commission for Cancer Research (92-37-CCR-00), a Sigma Xi Grant in Aidto G.R.S., and U.S.P.H.S. Grant AG09822 to M.V.H.

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the murine autologous mixed lymphocyte response: distribution of stimulator cells

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