The Murine Autologous Mixed Lymphocyte Response: Distribution of Stimulator Cells

Journal of Autoimmunity (1995) 8, 21–31

The Murine Autologous Mixed Lymphocyte Response: Distribution of Stimulator Cells

James E. Riggs, Gary R. Sirken, Lisa G. Prior and Monte 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 T cells proliferate when co-cultured with syngeneic, adult splenic B cells. Evidence suggests that expansion of such self-reactive T cells precedes their clonal deletion or functional inactivation (anergy). This mechanism may be of particular significance for establishing peripheral (extrathymic) tolerance. In order to assess the autostimulatory capacity of B cells present in a variety of lymphoid tissues, neonatal T cells were cultured with Peyer’s patch and peritoneal cavity cells. The results indicate that B cells in these tissues readily induce T cell proliferation. Evidence suggests that the B-1 B cell subpopulation is not obligatory for this process. Self-reactive T cells were evident in the spleen of young mice, substantiating a role for B cells in maintaining peripheral tolerance. The results suggest that B cells involved in this process are distributed in a variety of lymphoid organs.

Introduction Deletion and functional inactivation of self-reactive T lymphocytes are requisite to avert autoimmunity. Apparently, these processes are less efficient early in development 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 that autoreactive T cells are not restricted to the thymus (THY) [5, 6]. Peripheral (extrathymic) tolerance is essential for controlling these autoreactive escapees of clonal deletion [2]. The cells that induce peripheral tolerance, and their anatomical distribution, 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. 21 0896-8411/95/010021+11 $08.00/0

? 1995 Academic Press Limited

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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 that precedes tolerance [14, 15]. Consistent with this notion, the age at which stimulatory SP B cells become active in the murine AMLR correlates with the age at 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. A limited analysis of the tissue distribution of B cells stimulatory in the murine AMLR has been conducted [16, 17]. Lymphocytes from Peyer’s patches (PP) and the peritoneal 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 B cell sources and B cell subpopulations were tested. AMLR-stimulatory B cells were found equally distributed in SP, PerC, and PP, negating a specific role for the B-1 B cell subpopulation in the AMLR. These findings are discussed in the context of B 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 and C57BL/6J. mev (motheaten viable) mice (Ighb, H-2d, Mlsb), bred and maintained at Rider College, were used between 3 days and 32 weeks of age. MEV mice were derived from breeding pairs obtained from the Jackson Laboratory, Bar Harbor, ME. All other strains were derived from breeding pairs provided by Raquel Davis and Donald Mosier, The Scripps Research Institute, La Jolla, CA. All mice were housed and handled in accord with NIH guidelines. Cell preparations SP, PP, and THY cell suspensions were prepared by mincing between sterile glass slides in HBSS. RBCs were removed from SP cell suspensions by osmotic shock with Tris-buffered ammonium chloride [22]. PerC cells were collected by flushing the peritoneum with 10 ml of warm (37)C) HBSS. SP, PP, and PerC cell suspensions were T cell-depleted using HO-13-4 MAb (rat IgM anti-mouse Thy 1.2 [23]) plus baby rabbit complement (Pel-Freeze Biologicals, Rogers, AK) treatment followed by density gradient centrifugation (Lympholyte M, Accurate Chemical, NY) to retrieve viable cells. As total THY cell preparations enriched for adult T cells afford better MLRs [24], CD4 + 8 " thymocytes were enriched by depleting CD4 + 8 + and CD4 " 8 + T cells by two rounds of 3.155 MAb (rat IgM anti-mouse CD8 [25]) plus complement (Low Tox M, Accurate Chemical) treatment followed by density gradient centrifugation. RBC-depleted SP, PP, and

Distribution of AMLR-stimulatory B cells

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PerC cells were depleted of J11d + cells by two rounds of J11d MAb (rat IgM anti-mouse J11d [26]) plus baby rabbit complement treatment. To prepare peripheral CD4 + T cells, RBC-depleted SP cells were depleted of B and CD8 + T cells by two rounds of treatment with J11d and 3.155 MAbs plus baby rabbit complement treatment followed by density gradient centrifugation. The latter procedure routinely resulted in recovery of 27.5&1.5 (SE)% of input SP cells from 7-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 Technologies Inc., Grand Island, NY) supplemented with 10% heat-inactivated FCS (Hyclone, Logan, UT), 1 ìg/ml gentamycin, 100 u/ml penicillin, 100 ìg/ml streptomycin, 5#10 "5  2-mercaptoethanol, and 2 m L-glutamine. Responder T cells (2#105/well) were cultured with 4.0, 2.0, 1.0, 0.5, or 0.25#105 SP, PP, or PerC 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 at 37)C for 72 h. One ìCi/well of [3H]thymidine was then added and wells were harvested 17 h later with a cell harvester (Skatron Inst., Sterling, VA). Radioactivity was measured by scintillation spectrometry and results are expressed as counts per minute (cpm) &SEM for n=4 wells/sample tested. All T cell preparations were cultured with Mls-disparate DBA/2J stimulator cells as proliferation controls. As noted in the original description of the AMLR [4], experiments using mitomycin-C treated stimulator cells (SP, PP, or PerC) confirmed that T cells constitute the thymidine-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 that SP cells were more effective than lymph node, bone marrow, and THY cells for inducing T cell proliferation [13, 16]. B cell sources and distinct B cell subpopulations not studied in the murine AMLR include PerC cells, enriched for B-1 B cells, and PP cells, which lack the B-1 B cell subpopulation [4, 20, 29, 30]. The ability of these B cells sources to generate the AMLR were compared with that of SP cells. Both PP and PerC cells were stimulatory; however, PerC cells induced lower levels of neonatal T cell proliferation and were inhibitory when plated at a higher density (2#105 cells/well) (Figure 1). That adult T cells did not proliferate when co-cultured with any of the stimulator cell preparations is consistent with prior descriptions of the AMLR [3, 4, 13, 17]. Adult T cells are fully functional,

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Figure 1. Neonatal (/) and adult (.) T cell proliferation induced by SP, PP, and PerC cells from normal 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 are presented as the average CPM of a minimum of five cultures (n=4 wells/culture).

however, as they proliferate when cultured with Mls-disparate (DBA/2J) spleen cells (not shown). These results reveal that AMLR-stimulatory cells are distributed in 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 for generating the murine AMLR [13], they are likely to be the stimulatory population in PerC or PP preparations. To test this, PerC, SP, and PP cells from B cell-defective XID [31] and B and T cell-defective SCID [32] mice were employed as stimulator cells in the AMLR. As shown previously with SP cells [33], PP and PerC cells from XID mice fail to induce significant levels of neonatal T cell proliferation (Figure 2). SP and PerC cells from lymphocyte-deficient SCID mice also fail to generate the AMLR (PP are absent from SCID mice and thus were not tested). However, SP and PerC cells from XID and SCID mice reconstituted with normal (C.B-17 or BALB/c) B cells generate the AMLR (Figure 3). These results illustrate 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 cellreactive T cells are evident in the periphery of young mice, splenic CD4 + 8 " T cells

Distribution of AMLR-stimulatory B cells

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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 with 2#105 4–10-day-old BALB.xid CD4 + 8 " thymocytes. Results represent the average cpm of a minimum of three cultures (n=4 wells/culture).

Figure 3. Neonatal T cell proliferation induced by SP and PerC cells obtained from B cellreconstituted (/) XID (a) or SCID (b) mice. 2.0#105 SP and 1.0#105 PerC cells from XID or SCID mice, injected i.v. 8 weeks previously with 107 C.B-17 or BALB/c SP cells, were co-cultured with 2#105 4–8-day-old BALB.xid CD4 + 8 " thymocytes. Results represent the average cpm of a minimum of three cultures (n=4 wells/culture). Controls (.) represent age-matched, unreconstituted XID and SCID mice.

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

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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 were cultured 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 represent the average of four cultures (n=4 wells/culture). Excepting 2-week-old spleen, all T cell preparations exhibited >100,000 cpm when cultured with DBA/2J SP cells.

SP and PerC B cell-reactive T cells were evident in the thymi of 2-week-old mice and 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 T cells 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 were obtained using normal mice as donors of splenic T cells (data not shown). This result suggests that self-B cell reactive T cells may comprise a significant proportion of 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 would be an effective B cell subset for generating the AMLR. The lack of AMLR stimulation 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 the B-1 B cell subpopulation and generate the AMLR. B-1 cells alone, therefore, would appear to not be requisite for the AMLR. To define more rigorously the AMLRstimulatory capacity of B-1 B cells, SP cells from MEV mice were cultured with syngeneic, neonatal T cells. All B cells in mice homozygous for the mev gene have

Distribution of AMLR-stimulatory B cells

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Figure 5. IgM levels (a) found in and neonatal T cell proliferation (b) induced by C57BL/6J and C57BL/6J.mev mice. Serum IgM levels of 6–12-week-old mice were measured by ELISA. 2.0#105 SP cells 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).

been described as members of the B-1 subset [39]. Homozygotes exhibit characteristics ascribed to the B-1 subpopulation, e.g. high levels of spontaneous IgM production; heterozygotes have serum IgM levels similar to normal C57B L/6J mice (Figure 5(a)) [20, 39]. Compared with normal C57BL/6J and heterozygous C57BL/6J.mevv SP cells, homozygous C57BL/6J.mev SP cells are less effective in generating the AMLR (Figure 5(b)). The low AMLR found in these cultures was consistent with prior studies of the C57BL/6J and B-1-enriched NZB strains [16, 40]. These data suggest that B-1 B cells are not required to stimulate T cell proliferation. J11d " PerC cells induce neonatal T cell proliferation To investigate further the role of specific B cell subpopulations in the induction of T 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 " B cells are reported to represent the memory precursor and memory B cell pool [21]. SP and PP preparations depleted of J11d + cells had markedly reduced AMLRstimulatory capacity whereas J11d " PerC cells remained stimulatory (Figure 6(b)). B cell depletion was confirmed by the abrogation of LPS responsiveness (Figure 6(a)). These data reinforce the idea that B cells are the stimulatory subpopulation in the AMLR and reveal that J11d " PerC cells are stimulatory. Discussion By employing the murine AMLR to assess potential anatomic sites and cells involved in the maintenance of tolerance, AMLR-stimulatory cells were found in the SP, PerC and PP of normal, but not XID or SCID mice. B cells are the stimulatory cell type in these tissues as SP and PerC cells from B cell-reconstituted XID and SCID mice generate the murine AMLR. An obligatory role for B-1 B cells was negated by the failure of SP B cells from MEV mice, composed primarily of the

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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 cells were cultured at 2.0 (SP and PP) of 1.0#105 (PerC)/well with 25 ìg/ml LPS or 2#105 4–10-day-old BALB.xid 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 the AMLR. That autoreactive T cells were found in the spleen of young mice supports the hypothesis that the AMLR represents an interaction that precedes the acquisition of extrathymic tolerance. Combined, the data suggest that distribution of AMLR-stimulatory B cells throughout the immune system ensures the means whereby 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 not required to generate the AMLR. This was surprising because characteristics of B-1 B cells indicate that they could be potent in the AMLR: (1) prominence in the thymus [36], (2) absence from XID mice [38], and (3) specificity for autoantigens [30, 37]. Prior studies, however, had shown that NZB mice, noted for increased representation of the B-1 B cell subset [29], do not exhibit the AMLR [40]. Cells from C57BL/6 mice exhibit a meager AMLR [16, 39] that we have shown is further constrained by the mev mutation (Figure 5). Therefore, strain-specific variability and similar stimulatory capacity amongst SP, PerC and PP cells suggest that the pool of B cells that generates the AMLR does not have to include the B-1 subpopulation. Rather, collectively, these observations suggest that B-1 B cells may inhibit 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 allogeneic or Mls MLRs [41]. Activated B cells certainly are present in the SP, PP, and PerC of 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 activation is a phenotypic hallmark of XID mice and thus, a potential reason for their lacking the AMLR [31, 33, 42]. Furthermore, that xid B cells bind but fail to become activated by polyvalent antigens [31] may provide insight as to the nature of the self-antigen(s) being recognized in the AMLR.

Distribution of AMLR-stimulatory B cells

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One possible consequence of B cell activation is memory cell generation. B cells that enter this differentiation state are long-lived and lose an epitope recognized by the MAb J11d [26]. J11d " SP and PP cells failed to generate the AMLR whereas J11d " PerC did, indicating that the latter may represent a distinct subpopulation. This anatomic restriction is curious yet consistent with studies showing that peritoneal B cells are long-lived [29]. Prior studies have shown that maintenance of immunologic memory and tolerance requires the persistence of Ag and APC, respectively [43, 44]. Correlating these observations raises the possibility that adult T 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 which stimulatory B cells are first evident correlates with the age at which T cell responsiveness declines [16, 35] and more recent work showing that B cell acquisition of the ability to induce IL-2 production occurs late during the perinatal period [45]. In summary, we have shown that AMLR-stimulatory B cells, potentially responsible for maintenance of extrathymic tolerance, are distributed in a wide variety of lymphoid tissues. These cells may play a significant role in maintaining selftolerance in T cells that escape clonal deletion in the thymus. Abrogation of this form of tolerance may precede the onset of autoimmunity. Acknowledgements We thank Daniel Spector for maintenance of our mouse colony and Laura Blinderman for critical review of this manuscript. This work was supported by grants to J.E.R. from the NIH AREA program (R15 HD29206-01) and the New Jersey Commission for Cancer Research (92-37-CCR-00), a Sigma Xi Grant in Aid to G.R.S., and U.S.P.H.S. Grant AG09822 to M.V.H. References 1. Schneider, R., R. K. Lees, T. Pedrazzini, R. M. Zinkernagel, H. Hengartner, and H. R. MacDonald. 1989. Postnatal disappearance of self-reactive (Vâ6 + ) cells from the thymus of Mlsa mice. Implications for T cell development and autoimmunity. J. Exp. Med. 169: 2149–2158 2. Jones, L. A., L. T. Chin, G. R. Merriam, L. M. Nelson, and A. M. Kruisbeck. 1990. Failure of clonal deletion in neonatally thymectomized mice: tolerance is preserved through clonal anergy. J. Exp. Med. 172: 1277–1285 3. Howe, M. L., A. L. Goldstein, and J. R. Battisto. 1970. Isogeneic lymphocyte interaction: recognition of self antigens by cells of the neonatal thymus. Proc. Natl. Acad. Sci. USA 67: 613–619 4. Weksler, M. E., C. E. Moody, and R. W. Kozak. 1981. The autologous mixed lymphocyte reaction. Adv. Immunol. 31: 271–312 5. Smith, H., M. Chen, R. Kubo, and K. S. K. Tung. 1989. Neonatal thymectomy results in a repertoire enriched in T cells deleted in adult thymus. Science 245: 749–752 6. Sakaguchi, S. and N. Sakaguchi. 1990. Thymus and autoimmunity: capacity of the normal thymus to produce pathogenic self-reactive T cells and conditions required for their induction of autoimmune disease. J. Exp. Med. 172: 537–545 7. Ryan, J. J., R. E. Gross, K. S. Hathcock, and R. J. Hodes. 1984. Recognition and response to alloantigens in vivo. II. Priming with accessory cell-depleted donor

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