0014-2980/99/0909-2705$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999

Relative efficiency of microglia, astrocytes,dendritic cells and B cells in naive CD4+ T cellpriming and Th1/Th2 cell restimulation

Francesca Aloisi1, Francesco Ria2, Sandra Columba-Cabezas1, Henry Hess3,Giuseppe Penna3 and Luciano Adorini3

1 Laboratory of Organ and System Pathophysiology, Istituto Superiore di Sanita, Roma, Italy2 Institute of General Pathology, Catholic University, Roma, Italy3 Roche Milano Ricerche, Milano, Italy

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323–339-dependentactivation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells wasassessed in the presence of microglia and astrocytes from the neonatal mouse brain as wellas dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the mostefficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells,followed by IFN- + -preactivated microglia, large and small B cells. Astrocytes failed to acti-vate naive T cells. IFN- + -pretreated microglia were as efficient as DC in the restimulation ofTh1 cells, whereas IFN- + -pretreated astrocytes, large and small B cells were much less effi-cient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. Dur-ing T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 weresecreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC estab-lished in this study indicates that DC and microglia are the most efficient in the stimulation ofnaive CD4+ T cells and in the restimulation of Th1 cells, suggesting that activated microgliamay effectively contribute to Th1 responses leading to central nervous system inflammationand tissue damage. These potentially pathogenic responses could be counteracted by thehigh efficiency of astrocytes as well as microglia in restimulating Th2 cells.

Key words: Antigen presentation / Neural cell / Dendritic cell / B cell / IL-12

Received 25/1/99Revised 29/4/99Accepted 31/5/99

[I 19238]

Abbreviations: CNS: Central nervous system BBB: Blood-brain barrier DC: Dendritic cells MS: Multiple sclerosis

1 Introduction

CD4+ T cells are critical in the host resistance to neuro-tropic pathogens and in the development of central ner-vous system (CNS) autoimmunity [1]. Th1 cells secretingIL-2, IFN- + and TNF- g induce neuroinflammatoryresponses that have been implicated in the elimination ofneurotropic viruses and parasites, in virus-induced CNSimmunopathology and in the pathogenesis of EAE aswell as the human demyelinating disease multiple sclero-sis (MS) [2–4]. Th2-type cytokines (IL-4, IL-10) have alsobeen detected in the inflamed CNS [5, 6] and are thoughtto down-regulate Th1 responses [7, 8].

The current paradigm is that T cells primed in lymphoidorgans cross the blood-brain barrier (BBB) and arerestimulated within the CNS by the target Ag presentedby CNS APC. The initial inflammatory response facili-tates the recruitment and activation of additional T cells,leading to the spreading and chronicity of the immuneresponse [3]. Perivascular cells, a population of MHCclass II+ macrophage-like cells located within the basallamina of cerebral vessels, and microglia, bone marrow-derived cells residing in the CNS parenchyma proper, arethought to be implicated in the reactivation of CNS-infiltrating CD4+ T cells [9–11]. In the healthy CNS,microglia are ramified, non-phagocytic cells thatexpress low levels of macrophage markers (CD45,CD14, Fc receptors) and MHC class II molecules [11],and fail to restimulate T cells [10]. In EAE and MS, acti-vated microglia exhibit increased expression of MHCclass II molecules and up-regulate several adhesion/co-stimulatory molecules, including CD11a, CD40, CD54,CD80 and CD86, which mediate APC-T cell interaction

Eur. J. Immunol. 1999. 29: 2705–2714 T cell priming and restimulation by lymphoid and neural APC 2705

Figure 1. Relative efficiency of CNS and lymphoid APC in stimulating the proliferation of naive CD4+ T cells. Flow cytometricanalysis of lymph node and spleen cells from DO11.10 TCR-transgenic mice stained with anti-CD62(L-selectin), a naive T cell marker, and anti-CD4 mAb. (A) Total CD4+ T cells enriched from lymph nodes and spleen cells; aboutone-third express L-selectin. (B) Highly enriched CD4+ naive T cells, purified from the population in (A) after selection for highexpression of L-selectin. Values represent percent of positive cells. (C) Proliferation of naive CD4+ T cells. TCR-transgenic naiveCD4+ T cells (5 × 104) were cultured with graded numbers of APC (unstimulated or IFN- + -pretreated astrocytes or microglia, DC,large or small Bcells), in the presence of 0.3 ? M OVA 323–339. Proliferation was measured by incorporation of [3H]thymidine dur-ing the last 16 h of a 48-h coculture. Background cpm in the absence of Ag were 200–400. The data shown are means ± SD froma representative experiment run in duplicate, out of two performed. 1 , unstimulated astrocytes; ‡ , IFN- + -pretreated astrocytes;Æ , unstimulated microglia; , , IFN- + -pretreated microglia; Y , DC; ß , large B; | , small B.

and activation [11–15]. In vitro and in vivo activatedmicroglia can process and present Ag to T cell lines,leading to T cell proliferation [16, 17] and cytokine secre-tion [18]. Cultured rat or human microglia also primealloreactive CD4+ T cells [19, 20] and restimulate memoryT cells specific for viral antigens [21] or microorganismslike Candida albicans [20]. We have recently shown thatmouse microglia restimulate both Th1 and Th2 cells [22].After in vitro exposure to IFN- + and bacterial products[23, 24], upon interaction with Th1 cells [25], or duringthe Th1-mediated disease EAE [26], microglia produceIL-12, a critical cytokine for Th1 development and IFN- +secretion [27].

In CNS infection or immunopathology, astrocytes, themajor CNS glial cell population, are recruited into theinflammatory network [28] but only rarely express MHCclass II molecules ([29], and references therein). Asopposed to the in vivo situation, cultured astrocytesexpress MHC class II, CD54 and CD106 molecules whenexposed to IFN- + , IL-1 or TNF- § [29]. Conflicting dataexist on the capacity of astrocytes to prime and restimu-late T cell responses [19, 20, 29–32]. We have recentlyshown that astrocytes fail to express co-stimulatory mol-ecules (CD40, CD80, CD86) or produce IL-12, have aweak Ag processing capacity and present antigenic pep-tides mainly to Th2 cells [22, 23, 25].

APC recruited from the blood, such as macrophages,B cells and dendritic cells (DC), are also likely to play arole in the restimulation of effector T cells in the inflamedCNS [33–35]. To understand the relative contribution of

resident and recruited APC to intracerebral T cellresponses, it is important to establish a hierarchy amongthese cell types in Ag presentation. While this issue hasbeen extensively investigated for lymphoid APC [36, 37],the efficiency of CNS APC in the priming and restimula-tion of T cell responses is still poorly understood. In thepresent study we have used purified CD4+ naive T cellsas well as polarized Th1 and Th2 cell lines from DO11.10TCR-transgenic mice to directly compare the APC func-tion of microglia and astrocytes with that of DC, small(resting) and large (activated) B cells.

2 Results

2.1 DC and IFN- q -induced microglia are the mostefficient APC in the stimulation of naiveCD4+ T cells

DC expressing elevated levels of MHC class II, CD40,CD54 and CD80/CD86 molecules are the most potentAPC for T cell priming, whereas B cells expressing sur-face MHC class II, CD40 and CD54, but little CD80/CD86, are much less efficient [36, 37]. To evaluate theactual capacity of microglia and astrocytes to prime for Tcell responses, we have compared the efficiency of CNSand lymphoid APC in inducing the proliferation of OVATCR-transgenic naive CD4+ T cells enriched for highexpression of L-selectin (Fig. 1A, B). The APC function ofmicroglia and astrocytes was assessed after incubationfor 24 and 48 h, respectively, with or without 100 U/mlIFN- + . As previously shown [22], IFN- + -treated BALB/c

2706 F. Aloisi et al. Eur. J. Immunol. 1999. 29: 2705–2714

Figure 2. DC are the most efficient APC inducing IL-2 and IFN- + secretion by naive T cells. DO11.10 TCR-transgenic naive CD4+

T cells (5 × 104) were cultured together with IFN- + -pretreated astrocytes or microglia, DC and large or small B cells, in the pres-ence of 0.3 ? M OVA 323–339. After 24 and 72 h, culture supernatants from duplicate cultures were pooled and IL-2, IFN- + andIL-4 were quantified by specific ELISA. Values are means ± SEM from three experiments.

Figure 3. Induction of Th1 development in naive CD4+ cells by lymphoid and CNS APC. DO11.10 TCR-transgenic naive CD4+ Tcells (5 × 104) were cultured together with 3 × 104 IFN- + -pretreated astrocytes, microglia or DC and 3 × 105 large or small B cells,in the presence of 0.3 ? M OVA 323–339. After about 84 h, the cells were restimulated with PMA and ionomycin and stained forintracellular IFN- + and IL-4 as well as for surface CD4, as described in Sect. 4.7. Intracytoplasmic staining was assessed on CD4+

T cells. The experiment shown is representative of two performed. Values represent percent of positive cells.

mouse microglia express MHC class II (25–40 %), CD40(50–65 %) and CD54 (90–95 %), but neither CD80 norCD86 molecules. IFN- + -treated BALB/c mouse astro-cytes express MHC class II and CD54 (30–50 % and80–90 %, respectively), but no CD40, CD80 or CD86molecules [22].

In the presence of 0.3 ? M OVA 323–339, [3H]thymidineincorporation into naive CD4+ T cells was induced in anAPC density-dependent manner by DC, IFN- + -pretreated microglia, large and small B cells, but notby unstimulated microglia, unstimulated or IFN- + -pretreated astrocytes (Fig. 1C). DC are about threefoldmore efficient than IFN- + -pretreated microglia in stimu-lating naive T cell proliferation, whereas large and smallB cells are the least efficient.

The ability of CNS and lymphoid APC to stimulate naiveT cell proliferation correlated well with their ability toinduce IL-2 secretion. In the presence of 0.3 ? M OVA

323–339, DC (1 × 104 and 3 × 104/well) induced high IL-2secretion from naive CD4+ T cells, which was detectedafter 24 h of coculture and increased by about fourfold at72 h (Fig. 2). IFN- + -pretreated, but not unstimulated (notshown), microglia also induced IL-2 secretion from naiveT cells in a time- and cell density-dependent manner, butwere much less potent than DC. Compared to DC, largeand small B cells (3 × 105 cells/well) stimulated less IL-2secretion during T cell priming, whereas no IL-2 secre-tion was induced by unstimulated (not shown) or IFN- + -pretreated astrocytes (3 × 104 cells/well). Neither T cellproliferation nor IL-2 secretion was induced in theabsence of Ag or APC (not shown).

To compare the capacity of lymphoid and CNS APC topromote the maturation of naive CD4+ T cells into Th1 orTh2 cells, IFN- + and IL-4 production were measured byELISA and intracellular staining. In the presence of0.3 ? M OVA 323–339, DC were the most efficient APC ininducing IFN- + secretion by naive T cells in a time- and

Eur. J. Immunol. 1999. 29: 2705–2714 T cell priming and restimulation by lymphoid and neural APC 2707

Figure 4. Restimulation of Th1 and Th2 cells by CNS andlymphoid APC. Th1 and Th2 DO11.10 TCR-transgenicT cells (5 × 104) were cultured in the presence of 0.3 ? M OVA323–339 and graded numbers of APC. Astrocytes andmicroglia were preincubated for 24 h and 48 h, respectively,in the absence or presence of 100 U/ml IFN- + . After 24 h ofcoculture, supernatants from duplicate cultures were pooledand cytokines measured by two-site ELISA assays. Valuesare means ± SEM from three experiments. 1 , unstimulatedastrocytes; ‡ , IFN- + -treated astrocytes; Æ , unstimulatedmicroglia; , , IFN- + -treated microglia; Y , DC; ß , large B;| , small B.

cell density-dependent manner (Fig. 2). IFN- + -pretreatedmicroglia induced IFN- + secretion which was about onethird of that induced by similar numbers of DC. Thirty-and tenfold higher numbers of small and large B cellswere required to induce IFN- + secretion similar to thatinduced by DC and microglia, respectively, whereasastrocytes were ineffective. Neither IL-4 (Fig. 2) nor IL-10(not shown) were detected in the culture supernatantsunder any condition tested. No cytokine secretion wasdetected in the absence of Ag or APC (not shown).

Cytokine secretion data were consistent with intracellularstaining of CD4+ cells for IFN- + and IL-4 production (Fig.3).About 20 and 10% of naive CD4+ T cells produced IFN- +after priming with DC and IFN- + -pretreated microglia(3×104 cells/well), respectively. Large and small B cells(3×105 cells/well) induced 5% or less IFN- + -positiveT cells, whereas IFN- + -pretreated astrocytes (3×104 cells/well) were completely ineffective. No IL-4-producing cellswere induced by any of the APC tested.

2.2 Cytokine secretion by Th1 and Th2 cellsrestimulated by CNS and lymphoid APC

To determine the relative efficiency of CNS and lymphoidAPC in restimulating Th1 and Th2 cells, polarized CD4+

transgenic T cells were cultured with OVA 323–339(0.3 ? M) and graded numbers of APC. The degree of Tcellactivation was assessed by quantifying the amounts ofTh1 (IL-2 and IFN- + ) and Th2 (IL-4 and IL-10) cytokinessecreted in 24-h culture supernatants. TNF- § ,a cytokine secreted by both Th1 and Th2 cells [1], wasalso quantitated. DC and IFN- + -pretreated microglia weresimilarly efficient in stimulating the secretion of IL-2, IFN- +and TNF- § from Th1 cells, whereas unstimulated micro-glia induced little Th1 activation (Fig. 4). When stimulatedwith either APC, Th2 cells failed to secrete IL-2 or IFN- +and produced only low amounts of TNF- § . IFN- + -pretreated astrocytes and B cells were much less efficientthan DC and microglia in inducing cytokine secretion fromTh1 cells (Fig. 4). Ten- to 100-fold higher numbers ofastrocytes and B cells are required to induce Th1 cytokinesecretion similar to that induced by DC and IFN- + -pretreated microglia.

Less marked differences were observed in the capacityof CNS and lymphoid APC to restimulate Th2 cells. AllAPC types induced secretion of high amounts of IL-4from Th2 cells. DC, IFN- + -pretreated microglia andastrocytes were the most effective followed by large andsmall B cells. With respect to IL-10 secretion, IFN- + -pretreated microglia and DC were still the most potentAPC followed by IFN- + -pretreated astrocytes and Bcells.No cytokine secretion was detected in the absence of Agor with T cells or APC alone (not shown).

2.3 DC and IFN- q -induced microglia secretesimilar levels of IL-12 upon Ag-specificinteraction with Th1 cells, but DC secretemore IL-12 during priming of naive CD4+ cells

IL-12 plays a critical role in the differentiation of naiveTcells into Th1 and in the stimulation of Th1 proliferationand cytokine secretion [27]. As shown previously, DC[38] and microglia [25], but neither B cells [39] nor astro-

2708 F. Aloisi et al. Eur. J. Immunol. 1999. 29: 2705–2714

Figure 5. Secretion of IL-12 by CNS and lymphoid APC dur-ing T cell priming. DO11.10 TCR-transgenic CD4+ naiveT cells (5 × 104) were cultured in the presence of 0.3 ? M OVA323–339 and APC. Astrocytes and microglia were preincu-bated with 100 U/ml IFN- + for 24 h and 48 h, respectively.After 24 and 72 h of coculture, supernatants from duplicatecultures were pooled and IL-12 p40 was quantified by spe-cific ELISA. Values are means ± SEM from three experi-ments.

Figure 6. Microglia and DC produce similar amounts ofIL-12 upon interaction with Th1 cells. DO11.10 TCR-transgenic Th1 and Th2 cells (5 × 104) were cultured in thepresence of 0.3 ? M OVA 323–339 and microglia (eitherunstimulated or pretreated with IFN- + ) or DC. After 24 h,supernatants from duplicate cultures were pooled and IL-12p75 and p40 measured by specific ELISA. Values are means± SEM from three experiments.

cytes [25] secrete IL-12 upon Ag-specific interaction withTh1 cells. In the present study, we directly compared thecapacity of DC and microglia to secrete IL-12 during pre-sentation of OVA 323–339 to OVA TCR-transgenic naiveCD4+ as well as Th1 cells. As shown in Fig. 5, the amountof IL-12 p40 produced by DC during T cell priming wasmuch higher than that produced by IFN- + -pretreatedmicroglia. Astrocytes and B cells failed to secrete IL-12.Small amounts of IL-12 p75 (30 pg/ml) could be mea-sured only in 72-h supernatant of DC-naive T cell cocul-tures in only one out of four experiments performed. Theinability to detect IL-12 p75 during T cell priming may bedue either to low IL-12 production by DC or to its rapidconsumption by naive T cells during their maturation intoTh1. After 24-h coculture with Th1, but not Th2 cells,similar amounts of IL-12 p75 and p40 were secreted byIFN- + -pretreated microglia and DC (Fig. 6).

3 Discussion

We compared the ability of CNS and lymphoid APC toprime and restimulate CD4+ T cells from DO11.10 TCR-transgenic mice. Upon activation with IFN- + , culturedneonatal microglia can prime naive CD4+ T cells to prolif-erate and to develop into Th1 cells but are clearly lessefficient than DC. Conversely, activated microglia andDC display a similar efficiency in restimulating Th1 andTh2 cells and both APC types secrete IL-12 upon inter-action with Th1 cells. These findings suggest that micro-glia can contribute significantly to the reactivation of

T cells recruited to sites of CNS infection or inflamma-tion. The similar secretion of IL-12 by microglia and DCsupports a role for microglia in promoting Th1 cell activa-tion and CNS inflammation. In contrast, astrocytesresemble B cells in that both APC types are more effi-cient in restimulating Th2 rather than Th1 cells, fail tosecrete IL-12 and are inefficient in the priming of naiveCD4+ T cells.

In response to inflammatory stimuli, activated microgliaup-regulate expression of MHC class II and adhesion/co-stimulatory molecules [11–15, 20–22, 40]. Acquisi-tion of APC features may be particularly relevant in MSdemyelinating lesions, where phagocytosis of myelin byMHC class II+, CD40+, CD80/CD86+ activated microgliacould result in presentation of myelin Ag to autoreactiveCD4+ T cells secreting Th1-type cytokines [3]. AlthoughCD80/CD86-CD28 interactions play a critical role in Tcellpriming, they are also required for optimal cytokine pro-duction by effector T cells [41]. Ligation of CD40 on DCand macrophages by CD154 (CD40 ligand) expressed onT cells favors their maturation into efficient APC by up-regulating the expression of adhesion/co-stimulatorymolecules and by inducing the production of cytokines,including IL-12 [42, 43]. We have recently shown that theCD40/CD154 pathway is also important for IL-12 pro-

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duction by microglia during Ag presentation to Th1 cells[25]. This suggests a role for direct microglia-Th1 interac-tions in promoting microglia activation as well as a skew-ing of Th1 responses within the CNS.

Cultured mouse microglia analyzed in the present studywere ameboid, phagocytic cells obviously distinct fromresting, ramified microglia present in the normal adultCNS [10, 11, 16]. This may be due partly to their deriva-tion from the neonatal CNS (at this early time pointmicroglia are involved in the elimination of apoptoticcells occurring during brain development), and partly tothe fact that, before isolation, microglia are expanded invitro in the presence of astrocytes which secrete M-CSFand GM-CSF [28]. We assume that cultured neonatalmicroglia more closely resemble activated microglia thatdevelop in response to CNS injury of inflammation [11,40, 44]. By studying microglia acutely isolated from theCNS of normal adult rats and of rats undergoing GVHdisease, Sedgwick and collaborators [10, 18, 44] haveprovided evidence that microglia behave as efficientAPC only after they have acquired an activated,macrophage-like phenotype. While resting microgliaexpressing little if any MHC class II molecules fail to pre-sent myelin Ag to T cell lines [10], microglia activated byCNS-infiltrating T cells in GVH disease mature into MHCclass II-expressing, phagocytic cells which induce Th1-type cytokine secretion from T cell lines as efficiently asCNS-associated (perivascular, meningeal) macrophages[18, 44]. In preliminary experiments, microglia isolatedfrom the adult BALB/c mouse CNS and exposed for 24 hin vitro with IFN- + restimulate both Th1 and Th2 cells butare less potent than their neonatal counterparts and DC(R. De Simone and F. Aloisi, unpublished data). We arecurrently searching for additional molecules that maypromote microglia activation and APC function.

T cell priming within the CNS is unlikely to occur becauseonly activated T cells cross the BBB [45]. In CNS inflam-matory diseases, memory T cells are recruited to the CNSparenchyma [2, 4]. Some naive T cells could also gainaccess to the CNS after BBB disruption or via theinflamed endothelium [46]. The co-stimulatory require-ments for the reactivation of memory T cells are thoughtto be intermediate between those of naive and effectorT cells [46]. The present data clearly demonstrate thatIFN- + -pretreated mouse microglia exhibit T cell co-stimulatory potential. They are, however, less efficientthan DC in inducing proliferation and differentiation ofnaive CD4+ T cells into IFN- + -secreting Th1 cells. Thismay be due to the lack of CD80/CD86 molecules on IFN-+ -pretreated mouse microglia [22] as well as to the lowproduction of IL-12 by microglia during T cell priming. Wecannot exclude that the CD62-enriched T cell populationused in this study also contains a few memory T cells.

Even in this case, the present findings suggest that acti-vated microglia may contribute to the expansion andreactivation of recruited T cells to the CNS.

Cultured astrocytes, which also share several features ofreactive adult astrocytes [28], are much less potent thanmicroglia and DC in restimulating Th1 cells and fail toproduce IL-12 [22–25]. However, in the presence ofappropriate peptides, astrocytes stimulate cytokinesecretion from Th2 cells as efficiently as microglia andDC. In this respect, astrocytes behave similarly to B cellswhich, relative to DC, are less efficient stimulators of Th1responses. The low efficiency of astrocytes in Ag pro-cessing [22] coupled with the observation that MHCclass II+ astrocytes are rare in vivo [29], even duringchronic CNS inflammation, supports the view that astro-cytes have a marginal role as APC. In agreement withprevious reports showing that astrocytes fail to sustainthe proliferation of alloreactive CD4+ T cells [19, 20, 31],we have demonstrated that astrocytes are unable toprime naive CD4+ T cells. This finding contrasts with arecent study showing that IFN- + -activated astrocytesfrom the BALB/c mouse brain stimulate the proliferationof DO11.10 TCR-transgenic naive T cells [32]. Theunconfirmed findings of the latter study could beexplained by the use of non-purified naive T cells andcontamination of astrocyte cultures by microglia.

In conclusion, our comparative study indicates a hierar-chy of APC in which DC and, to a lesser extent, IFN- + -induced microglia are the most efficient in the priming ofnaive CD4+ T cells. Microglia and DC are also the mostefficient APC for the restimulation of Th1 cells followedby astrocytes and B cells. Conversely, differencesbetween the various APC examined are much lessmarked in the restimulation of Th2 cells, suggesting thatthe requirements for Th2 activation are less stringentthan for Th1 cells. The similarity between microglia andDC in stimulating Th1 responses as well as in IL-12secretion indicates that microglia activation may be rele-vant for the onset and severity of pro-inflammatory Th1responses in infectious and autoimmune CNS diseases.Conversely, the high efficiency of both microglia andastrocytes in restimulating Th2 responses could contrib-ute to up-regulate the intracerebral synthesis of anti-inflammatory cytokines, thus providing a local regulatorycircuit for the inhibition of Th1 responses.

4 Materials and methods

4.1 Mice

BALB/c mice were purchased from Charles River (Calco,Italy). DO11.10 TCR-transgenic mice on BALB/c back-ground [47] were kindly provided by Dr. D. Y. Loh. In these

2710 F. Aloisi et al. Eur. J. Immunol. 1999. 29: 2705–2714

transgenic mice, 95–100 % of the CD4+ T cells are V g 8.1.2+

[48] and express an § g TCR specific for OVA peptide323–339 bound to I-Ad.

4.2 Glial cell cultures

Cultures highly enriched in microglia (92–95 % Mac-1+ cells)and astrocytes (95–98 % glial fibrillary acidic protein+ cells)were established from the forebrains of 1-day-old BALB/cmice as previously described [22, 23].

4.3 DC, large and small B cell preparation

DC, large and small B cells were enriched from inguinallymph nodes of 2- to 3-month-old mice immunized with CFAinto the hind footpad 6 days earlier, as previously described[49]. Briefly, lymph node cells were depleted of T cells bycytotoxic elimination with HO-13-4 anti-Thy 1.2 antibody(TIB 99) followed by rabbit complement. Thy 1.2-negativecells were separated by Percoll gradient in low buoyant andhigh buoyant density cells. Small B cells were enriched fromhigh buoyant density cells by incubation with anti-CD45R(B220)-coated magnetic microbeads (Miltenyi-BiotecGmbH, Bergisch Gladbach, Germany) and the B220+ frac-tion was collected from MiniMACS separation columns( G 95 % B220+ cells). DC were purified from low buoyantdensity cells by using anti-CD11c (N418)-coated micro-beads. After two rounds of elution on MiniMACS separationcolumns, a highly enriched population of DC (85–95 %N418+ cells) was obtained. Large B cells were obtained fromlow buoyant, N418-negative cells, after incubation withB220-coated microbeads and two rounds of elution on Mini-MACS separation columns ( G 95 % B220+ cells). As previ-ously shown [50], the APC capacity of DC was not affectedby positive selection with N418 mAb.

4.4 Naive TCR-transgenic T cells

Naive CD4+ CD62 (L-selectin)high double-positive T cellswere selected from spleen and lymph nodes of DO11.10TCR-transgenic mice using FITC-conjugated anti-CD4(PharMingen, San Diego, CA) and MACS anti-FITC Multisortkit (Miltenyi-Biotec GmbH). After release of the magneticlabel, positive selection for CD62highcells was performed withanti-CD62 (MEL-14)-coated magnetic microbeads. Naive Tcells were stained by incubation with optimal concentrationsof FITC-conjugated anti-CD4 and PE-conjugated anti-CD62(PharMingen). Naive T cell populations routinely containedG 98 % CD4+ CD62high double-positive T cells.

4.5 Th1 and Th2 TCR-transgenic cell lines

CD4+ cells were positively selected from inguinal and mes-enteric lymph nodes of naive DO11.10 TCR-transgenic mice

by anti-CD4-coated magnetic microbeads (Miltenyi-BiotecGmbH). CD4+ T cells (2 × 105 cells/well) were cultured withOVA peptide 323–339 (0.3 ? M) and mitomycin c-treatedBALB/c splenocytes (5 × 106 cells/well) as APC in a total vol-ume of 2 ml in 24-well plates, in the presence of either0.1 ng/ml recombinant mouse IL-12 (Hoffmann-La RocheInc., Nutley, NJ) and 10 ? g/ml anti-mouse IL-4 (11B11,ATCC), or 20 ng/ml mouse recombinant IL-4 (Hoffmann-LaRoche AG, Basel, Switzerland) and 10 ? g/ml anti-mouseIL-12 mAb (10F6, Hoffmann-La Roche Inc.), to obtain Th1 orTh2 cell lines, respectively. Cells were cultured in RPMI sup-plemented with 50 ? M 2-ME, 2 mM L-glutamine, 50 ? g/mlgentamicin and 10 % FCS (Sigma, St. Louis, MO). After3days in vitro, T cells were expanded and grown in completemedium containing 10 ng/ml recombinant human IL-2(Hoffmann-La Roche AG). The phenotype of Th1 and Th2cell lines was determined by intracytoplasmic staining forIFN- + and IL-4, as previously described [38].

4.6 T cell stimulation assays

Varying numbers of APC were seeded in 96-well flat-bottomtissue culture plates. Prior to addition of T cells from trans-genic mice, microglia and astrocytes were treated for 24 and48 h, respectively, with medium (DMEM/10 % FCS) with orwithout recombinant mouse IFN- + (Genzyme, Cambridge,MA). TCR-transgenic Th1 and Th2 cells collected from 6- or7-day-old cultures or freshly prepared TCR-transgenic CD4+

naive T cells were added (5 × 104) in RPMI/10 % FCS (sup-plemented as described above) to wells containing the dif-ferent APC in the presence of a concentration of OVA323–339 (0.3 ? M) inducing approximately half-maximalT cell activation [48]. For analysis of secreted cytokines,supernatants from duplicate or triplicate cultures were har-vested after 24–96 h, centrifuged at 1200 rpm and stored at–20 °C until used for cytokine determination. For prolifera-tion assays of naive T cells, astrocytes and microglia, but notDC, large B or small B cells, were irradiated (3000 rad)immediately before addition of T cells and Ag. Proliferationwas measured by incorporation of 0.5–1 ? Ci/well [3H]thymi-dine (Amersham Italia, Milan, Italy; specific activity 25 Ci/mmol) during the last 16 h of a 48-h incubation period.Radioactivity was detected using a Packard Topcountmicroplate scintillation counter (Packard Instrument Co.,Meriden, CT).

IFN- + , TNF- § , IL-2, IL-4, IL-10, IL-12 p75 and total IL-12(mainly p40) were quantified by specific ELISA from Gen-zyme. Detection limits for all cytokines were in the range of5 to 15 pg/ml.

4.7 Intracellular staining for IFN- q and IL-4 production

Following interaction with APC in the presence of OVA323–339 peptide, T cells were restimulated with 50ng/ml PMAand 0.75 ? g/ml ionomycin for 4h at 37°C, with 10 ? g/ml

Eur. J. Immunol. 1999. 29: 2705–2714 T cell priming and restimulation by lymphoid and neural APC 2711

brefeldin A (Novartis Ltd., Basel, Switzerland) added for thelast 2 h. After fixing for 20 min at room temperature in 2 %formaldehyde, the cells were stained for intracytoplasmicIFN- + and IL-4 using the method described by Openshaw etal. [51]. All incubations were performed at room temperaturein PBS containing 5 % FCS, 0.5 % saponin (Sigma) and0.1 % sodium-azide. After washing and 10 min preincuba-tion in PBS/FCS/saponin, cells were incubated with FITC-conjugated rat anti-mouse IFN- + (XMG1.2, PharMingen) andPE-conjugated rat anti-mouse IL-4 (11B11, PharMingen) orwith FITC- and PE-labeled rat IgG1 isotype controls (R3-34,PharMingen). After 30 min, cells were washed twice withPBS/FCS/saponin and then with PBS/5 % FCS to allowmembrane closure. Cell membranes were then stained withCy-Chrome-labeled anti-CD4 (L3T4, PharMingen) for15 min. Analysis was performed with a FACScan® flow cyto-meter (Becton Dickinson and Co., Mountain View, CA)equipped with CELLQuest software, and 30 000 events wereacquired.

Acknowledgments: This research was supported in partby the Project on Multiple Sclerosis of the Istituto Superioredi Sanita/Italian Ministry of Health.

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Correspondence: Francesca Aloisi, Laboratory of Organand System Pathophysiology, Istituto Superiore di Sanita,Viale Regina Elena 299, I-00161 Roma, ItalyFax: +39-06-4957821e-mail: fos4 — iss.it

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