Phenotypic and Functional Characterization of Mouse Hepatic CD8α⁺ Lymphoid-Related Dendritic Cells

Animals

Male C57BL/10J (B10; H2^b) and C3H/HeJ (C3H; H2^k) mice, 8–12 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). They were housed in the specific pathogen-free facility of the University of Pittsburgh Medical Center and were provided with Purina rodent chow (Ralston Purina, St. Louis, MO) and tap water ad libitum.

Reagents

FITC-, PE-, or Cy-Chrome-conjugated mAbs to detect cell surface CD3ε, CD8α, CD8β, CD11b, CD11c, CD40, CD80, CD86, and IA^b β-chain expression by flow cytometry were purchased from PharMingen (San Diego, CA). Purified rat anti-CD8α (53-6.7), hamster anti-CD11c (HL3), biotin-conjugated hamster anti-CD3ε (145-2C11), and mouse anti-IA^b β-chain (25-9-17) mAb (PharMingen) were used for immunostaining of cytospins and tissue sections. Recombinant mouse GM-CSF was provided by Dr. S. K. Narula (Schering-Plough, Kenilworth, NJ). Chinese hamster ovary cell-derived human rFlt3L was provided by the Immunex Research and Development Corp. (Seattle, WA). RPMI 1640 (Life Technologies, Gaithersburg, MD) was supplemented with 10% FCS (Nalgene, Miami, FL), nonessential amino acids, l-glutamine, sodium pyruvate, penicillin-streptomycin, and 2-ME (all from Life Technologies) and is referred to subsequently as complete medium.

DC isolation and sorting

DC were isolated from the livers of animals given Flt3L (10 μg mouse/day i.p. in HBSS) for 6–10 consecutive days. Liver NPC were isolated as described previously (30), with the following modifications. Briefly, the liver was perfused in situ with 30 ml of HBSS, followed by 5 ml of type IV collagenase (1 mg/ml; Sigma) in HBSS. The liver was then removed, teased apart, and digested for an additional 30 min with 20 ml of the collagenase solution at 37°C. The resulting mixture was resuspended with a Pasteur pipette, and hepatocytes were sedimented by centrifugation at 40 × g. The NPC were recovered from the supernatant and further purified by density centrifugation using 30% metrizamide at 1200 × g for 20 min at 4°C. NPC were incubated overnight (18 h) in RPMI 1640 complete medium containing GM-CSF (4 ng/ml), unless otherwise stipulated. DC were enriched by metrizamide (16%) density centrifugation at 500 × g, for 10 min at room temperature (20°C). The buffy layer was aspirated, washed, and then labeled with anti-CD11c-FITC and either anti-CD8α-PE or anti-CD11b-PE. CD8α⁺ CD11c⁺ or CD8α⁻ CD11c⁺ DC populations, with high forward and side scatter profiles, were sorted using a Coulter EPICS Elite (Hialeah, FL) to >95% purity.

Morphologic characterization of liver DC subsets

Cytospins, prepared from bulk, overnight-cultured NPC, were air-dried and fixed with acetone for 5 min. Specimens were washed in HBSS, then incubated with a 1/50 dilution of rat anti-CD8α mAb overnight (18 h) at 4°C, followed by a 1/100 dilution of biotinylated goat anti-rat polyclonal Ab for 30 min at room temperature. Samples were then incubated with avidin-biotin complex/peroxidase (ABC-Px, Vector, Burlingame, CA) for 30 min at room temperature, and Px activity was detected with 3,3′-diaminobenzidine (Sigma) substrate kit. Specimens were counterstained lightly with hematoxylin (VWR Scientific, West Chester, PA). Cells stained with species-specific, isotype-matched Ig were used as negative controls. Sorted CD8α⁺ DC were examined ultrastructually by transmission electron microscopy. Briefly, cells were gently pelleted (500 × g) and fixed with 2.5% glutaraldehyde in PBS overnight at 4°C. Secondary fixation was conducted in 1% osmium tetroxide/1% Fe₆Cn₃ for 1 h, followed by dehydration through a graded ethanol series (30–100%). Pellets were then embedded in Polybed 812, and ultrathin sections (60 nm) were obtained using a Reichert Ultracut E microtome (Vienna, Austria). Sections were poststained in 2% uranyl acetate in 50% methanol for 10 min, and 1% lead citrate for 7 min and viewed on a JEOL JEM 1210 electron microscope (Peabody, MA) at 80 kV.

Flow cytometric analyses

Cell surface Ag immunostaining was performed as described previously (30). Leukocytes were first blocked with 10% normal goat serum for 20 min at 4°C and then stained with mAb for 30 min at 4°C. Cells stained with the appropriate isotype-matched Ig (PharMingen) were used as negative controls. After staining, the cells were fixed with 2% paraformaldehyde and analyzed using a Coulter Elite flow cytometer.

RT-PCR

Total cellular RNA was extracted with TRIzol (Life Technologies), and residual genomic DNA contamination was eliminated by DNase digestion (Life Technologies). First-strand cDNA was synthesized using Advantage RT-for-PCR (Clontech, Palo Alto, CA) with the supplied oligo(dT)₁₆ primer. Fifty to 100 ng of cDNA was used for each subsequent PCR. Relatively equal quantities of cDNA were ensured by amplifying a 540-bp segment of β-actin as described previously (31). FasL cDNA was detected by amplification of a 201-bp fragment (32). Conditions for PCR were 35 cycles of amplification and consisted of denaturation at 95°C for 20 s, 30-s annealing at 62 or 58°C for β-actin and FasL, respectively, and extension at 72°C for 30 s using a Gene Amp PCR System 2400 (Perkin-Elmer, Norwalk, CN). Amplified products were separated in a 1.5% agarose gel and visualized with ethidium bromide fluorescence.

In vivo trafficking of DC

Sorted, overnight-cultured CD8α⁺ or CD8α⁻ DC were washed extensively in HBSS, then injected s.c. (1–2.5 × 10⁵ in 50 μl) into the footpad or flank of one hindlimb. The regional lymph nodes (popliteal and inguinal), spleen, and thymus were removed after 2 days for immunohistochemical and molecular analyses and after 7 days for functional studies (lymph nodes only). Trafficking DC were identified by staining for donor MHC class II (IA^b; see below) and by detection of genomic DNA.

Mixed leukocyte reaction

Single-cell preparations from lymph nodes or spleens of C3H mice were used as responders. Splenocytes were enriched for T cells by a single passage through nylon wool columns (45 min; 37°C). Cells (2 × 10⁵) were placed in each well of 96-well, round-bottom plates, and varying numbers of gamma-irradiated (20 Gy), sorted, overnight-cultured CD8α⁺ or CD8α⁻ DC or normal bulk B10 splenocytes were added as stimulators. The cultures were incubated in RPMI 1640 complete medium for 72 h in a humidified atmosphere of 5% CO₂ in air. [³H]TdR (1 μCi in 10 μl) was added to each well for the final 18 h of culture. Cells were harvested using a multiple well harvester, and [³H]TdR incorporation was determined in a liquid scintillation counter. Results were expressed as the mean counts per minute ± 1 SD.

Immunostaining of tissue sections

Tissue samples were embedded in Tissue-Tek OCT (Miles Laboratories, Elkhart, IN), snap-frozen in isopentane (prechilled in liquid nitrogen), and stored at −80°C until use. Eight-micron cryostat sections were mounted on slides treated with Vectabond (Vector), air-dried, and fixed in cold acetone (4°C) for 10 min. Tissue sections were blocked with 5% goat serum, followed by avidin blocking solution (Vector). For simultaneous detection of CD8α and CD11c in normal liver, tissue sections were incubated successively with 1) rat anti-mouse CD8α mAb (1/100, 1 h at room temperature), 2) biotin polyclonal goat anti-rat Igs (1/100, 30 min at room temperature; PharMingen), and 3) ABC/alkaline phosphatase (AP; 30 min at room temperature; Vector). Following detection of AP activity with the substrate Vector Blue (Vector), the sections were further incubated with avidin blocking solution, and then stained successively with 1) hamster anti-mouse CD11c mAb (1/100, 1 h at room temperature), 2) biotin mouse anti-hamster IgG mAb (1/100, 1 h at room temperature; PharMingen), and 3) ABC-Px (30 min at room temperature). Px activity was detected with 3,3′-diaminobenzidine. For simultaneous detection of IA^b+ DC in T cell areas of recipient lymphoid tissues, sections were blocked as described, and CD8α⁺ DC were labeled with a 1/100 dilution of biotin-conjugated mouse anti-IA^b β-chain mAb, followed by the second-step reagent ABC-AP. Following detection of AP activity with the substrate Vector Blue, the sections were further incubated with avidin blocking solution, then stained with a 1/50 dilution of biotin-conjugated hamster anti-mouse CD3ε mAb, followed by ABC-Px. Px activity was detected with the 3-amino-9-ethylcarbazole substrate kit (Vector). Endogenous AP activity was inhibited by incorporation of levamisole (Vector) in the substrate solution. Endogenous Px activity was blocked by successive passages in 70% ethanol, 1% H₂O₂ in methanol, and 70% ethanol. Sections were mounted first in Crystal/Mount (Biomeda, Foster City, CA), then in Permount (Fisher Scientific, Pittsburgh, PA). Hamster or mouse isotype-matched Ig was used as the control.

Genomic PCR

Genomic DNA was extracted as described previously (33) with the following modifications. Tissue samples were digested overnight in lysis buffer (0.1 M Tris (pH 8.50, 100 mM NaCl, 5 mM EDTA, and 0.2% SDS) containing proteinase K (1 mg/ml) at 55°C. Genomic DNA was extracted with Tris-EDTA-saturated phenol/chloroform/isoamyl alcohol (25/24/1), followed by precipitation with 3 M sodium acetate (pH 5.2) in isopropyl alcohol. Excess salt was removed by washing with 70% ethanol. DNA was air-dried briefly and resuspended in Tris-EDTA. One microgram of genomic DNA was used for each PCR, and relatively equal quantities were ensured by amplifying β-actin as described for RT-PCR above. Donor-specific signal was detected by amplification of donor MHC class II (IA^b) DNA in the C3H recipients as previously described (31). Genomic DNA from B10 (0.1 μg in 0.9 μg of C3H) and C3H splenocytes were used as positive and negative controls, respectively.

CD8α⁺ and CD11b⁺ (CD8α⁻) DC in normal liver and kinetics of Flt3L-induced DC generation

The frequency of both CD8α⁺ and CD11b⁺ (CD8α⁻) DC in freshly isolated, normal liver NPC (day 0) was determined to be very low (≤1%; Fig. 1⇓A), which represents the lower limit of accurate flow cytometric detection. To convincingly demonstrate the presence of both DC populations in normal liver, well-established protocols for DC enrichment (overnight incubation of the bulk leukocyte population, followed by metrizamide density centrifugation) were used. Both CD8α⁺ and CD11b⁺ (CD8α⁻) DC were substantially enriched to 12–14% of the low buoyant density leukocyte fraction (Fig. 1⇓A), thus confirming that each subset was present in normal liver and in approximately equivalent numbers. To determine the time course of Flt3L-mediated induction of CD8α⁺ and CD11b⁺ (CD8α⁻) DC in the liver, mice were injected with 10 μg of Flt3L once daily for 6–10 days, a regimen previously described for the optimal induction of DC subsets in mouse lymphoid and nonlymphoid tissues (17, 34). At the end of each treatment period, NPC were isolated as described in Materials and Methods, immunolabeled, and analyzed by flow cytometry. Flt3L administration substantially increased the frequency of CD11c⁺ cells to a maximum of 55–60% of the total NPC population, which was maintained from days 8–10 of treatment (Fig. 1⇓A). Similar increases were observed in the CD8α⁺ (29%; day 10) and CD11b⁺ (27%) DC subsets. Flt3L increased the total number of DC per liver most dramatically, with the maximal increase of 650-fold observed after 10 days of treatment. The progressive increase in total CD8α⁺ and CD11b⁺ DC in the liver is shown in Fig. 1⇓B. Both DC subsets were increased relatively equally by Flt3L. CD8α⁺ DC could readily be identified in other tissues of Flt3L-treated animals, including bone marrow, spleen, and thymus (Fig. 2⇓). Although the total number of leukocytes in bone marrow and thymus was not altered substantially by Flt3L administration, small increases in CD8α⁺ DC frequency were noted. In contrast, the total number of splenocytes was increased 5-fold (data not shown), and the CD8α⁺ DC frequency was increased 15-fold by Flt3L administration (Fig. 2⇓). These data regarding bone marrow, thymus, and spleen are consistent with previous reports (17, 35).

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FIGURE 1.

Detection of CD8α⁺ and CD8α⁻ DC in normal liver and Flt3L-induced changes in the incidence and absolute number of each. DC were enriched from normal B10 liver by overnight culture of NPC followed by metrizamide density centrifugation. B10 mice were treated daily with Flt3L, as described in Materials and Methods. Total numbers of leukocytes per liver were enumerated. Freshly isolated NPC were double stained with FITC-conjugated anti-CD8α or anti-CD11b mAb together with anti-CD11c PE mAb and then analyzed by flow cytometry. The time course of Flt3L-induced increases in CD8α⁺ and CD11b⁺ (CD8α⁻) DC in liver is shown (A). Based on the absolute number of leukocytes per organ and the percentage of mAb-positive cells, the total numbers of CD8α⁺ CD11c⁺ (○) or CD11b⁺ CD11c⁺ DC (•) were calculated for each time point and plotted (B). The results are representative of two separate experiments.

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FIGURE 2.

Flt3L-induced increases, concomitant with those in liver, in the incidence of CD8α⁺ DC in hemopoietic and lymphoid tissues. B10 mice were treated for 10 days with Flt3L as described in Materials and Methods. Freshly isolated leukocytes were double stained with anti-CD8α FITC and anti-CD11c PE mAb and analyzed by flow cytometry. The increase in CD8α⁺ DC incidence after 10 days of Flt3L administration is shown for bone marrow, spleen, and thymus. The results are representative of two separate experiments.

Morphologic appearance of liver CD8α⁺ DC

Double immunostaining to detect coexpression of CD8α and CD11c (LDC), identified a small number of DC-like single cells in the portal spaces and the perivenular regions of normal mouse livers (Fig. 3⇓A). Other cells with dendritic morphology but CD8α⁻ CD11c⁺ (myeloid DC), and small round CD8α⁺ CD11c⁻ cells (presumed CD8 T cells) were also detected (Fig. 3⇓A). CD8α⁺ CD11c⁺ liver DC induced by Flt3L were examined morphologically both in cytocentrifuge preparations and by transmission electron microscopy of flow-sorted specimens. As shown in Fig. 3⇓, B–D, these cells exhibited typical reniform or lobulated nuclei, few prominent granules or vesicles, and extensive cell membrane projections characteristic of DC, including liver-derived MDC (30). Birbeck granules were not observed.

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FIGURE 3.

Characteristics of liver lymphoid DC (LDC). A, Distribution of CD8α⁺ DC in normal liver. Tissue sections were double labeled to detect CD8α (blue) and CD11c (brown). CD8α⁺ cells with dendriform appearance (black arrows and inset to show apparent dual expression of CD8α and CD11c) are located mainly in portal spaces (the vein lumen is marked with an asterisk). Two CD8α⁻ CD11c⁺ DC (MDC) are indicated by arrowheads at the top right. The red arrow marks a CD8α⁺ CD11c⁻ small round cell (presumed CD8⁺ T lymphocyte). ABC-Px/ABC-AP; original magnification, ×40. B and C, Cytospin preparations of bulk, overnight-cultured liver NPC stained with Giemsa (B) and anti-CD8α mAb (C; brown; ABC-Px). The dendriform facies of CD8α⁺ DC is readily apparent. Original magnification, ×100. D, Transmission electron micrograph of flow-sorted, CD8α⁺ CD11c⁺ hepatic DC (bar at bottom right = 5 μm).

Phenotype and purification of liver-derived DC subsets

Consistent with previous reports (12) regarding thymic and splenic DC, both freshly isolated and overnight-incubated, hepatic CD8α⁺ DC expressed low levels of CD11b (Fig. 4⇓A), in contrast to the high levels detected on CD8α⁻ DC (Fig. 4⇓B). There was a broad gradation of CD8α expression by DC, and the mean fluorescence intensity of CD8α expression was generally less on DC compared with that on CD11c⁻ CD8α⁺ hepatic T cells (Figs. 1⇑A and 5A). Interestingly, a small (14–20%) subset of CD8α⁺ DC coexpressed low levels of surface CD8β (Fig. 4⇓C). Vremec et al. (12) reported low levels of expression of the CD8αβ heterodimer by thymic DC, indicating that the αα homodimer was the main form of CD8 expression on LDC.

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FIGURE 4.

Flow cytometric analysis of sorted hepatic DC demonstrates that CD8α⁺ CD11c⁺ DC (A) express low levels of the myeloid-associated Ag CD11b compared with the relatively high levels expressed by CD8α⁻ CD11c⁺ DC (B). A minor subpopulation of CD8α⁺ CD11c⁺ DC exhibited positive staining for CD8β (C). Dotted profiles show isotype-matched control values. mfi, mean fluorescence intensity. Data are representative of three separate experiments.

To further examine the surface Ag phenotype of DC subsets isolated from Flt3L-treated mouse liver, NPC were triple immunostained, both when freshly isolated and following overnight (18-h) incubation in the presence of GM-CSF. GM-CSF has been reported to improve bulk DC viability and recovery in short term splenocyte cultures (11). Inclusion of GM-CSF during overnight incubation improved the survival and recovery of NPC, assessed by uptake of the nonvital stains PI or trypan blue with detection by flow cytometry or direct cells counts, respectively. Typically, NPC viability and recovery were improved 12–25% by inclusion of GM-CSF during overnight culture (data not shown).

In freshly isolated liver NPC, both DC subpopulations harvested after 10 days of Flt3L administration expressed a surface phenotype of immature DC (Fig. 5⇓A). Thus, low levels of expression of MHC class II (IA^b), CD80, and CD86 were detected, while staining for CD40 was low/negative. Interestingly, CD8α⁺ DC from freshly isolated NPC expressed consistently higher levels of MHC class II, CD80, and CD86 (four experiments), with a minor population (∼10%) of CD40⁺ cells. By contrast, after overnight culture in GM-CSF, both CD8α⁺ and CD8α⁻ DC markedly up-regulated expression of these Ags to become MHC class II^high, CD40⁺, CD80^high, and CD86^high (Fig. 5⇓B). The expression of MHC class II and costimulatory molecules on both subsets cultured overnight was substantially reduced in the absence of GM-CSF (Table I⇓). However, the relative levels of MHC class II and costimulatory molecule expression between CD8α⁺ and CD8α⁻ DC subsets were similar in both the presence and the absence of GM-CSF. In subsequent experiments CD8α⁺ and CD8α⁻ DCsubsets were sorted using gates to exclude CD11c^dim, low forward/side scatter ratio cells, which generally included a high proportion of dead or dying cells.

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FIGURE 5.

Flow cytometric analysis of liver NPC from B10 mice after 10 days of Flt3L administration. NPC were triple immunolabeled with 1) anti-CD11c PE, 2) anti-CD8α Cy-Chrome, and 3) anti-MHC class II (IA^b), anti-CD40, anti-CD80, or anti-CD86 -FITC. Scatter plots depict CD8α⁺ and CD8α⁻ DC populations in freshly isolated (A) and overnight-cultured (B) NPC. Shaded profiles indicate the expression of specific markers (FITC) on freshly isolated and overnight-cultured CD8α⁺ and CD8α⁻ DC subpopulations gated as shown. The data demonstrate that culture of freshly isolated, immature liver DC results in phenotypic maturation of both DC subsets. Open profiles show isotype-matched controls. The results are representative of four separate experiments.

RT-PCR analysis of CD95L (FasL) expression

The expression of gene transcripts for CD95L in both freshly isolated and overnight-cultured, sorted CD8α⁺ and CD8α⁻ DC from the liver was identified using PCR (Fig. 6⇓). In two separate experiments, transcripts for CD95L were identified in all DC subpopulations, with the highest level detected in freshly isolated, immature CD8α⁻ DC in both experiments. Expression of cell surface CD95L protein was not detected by mAb staining and flow cytometry on either freshly isolated or overnight-cultured CD8α⁺ or CD8α⁻ DC (data not shown).

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FIGURE 6.

RT-PCR analysis of CD95L (FasL) mRNA expression in normal B10 mouse kidney (KID; low expression), 48-h Con A-activated B10 splenocytes (SPL; high expression), and both freshly isolated immature (i), and overnight-cultured mature (m), sorted liver CD8α⁺ and CD8α⁻ DC. Amplification of β-actin was used as the internal positive control. Data are representative of two separate experiments.

Comparative allostimulatory activity of sorted CD8α⁺ and CD8α⁻ DC in vitro

The ability of sorted, overnight-cultured B10 CD8α⁺ and CD8α⁻ DC to stimulate the proliferation of naive allogeneic (C3H) T cells was assessed initially in vitro. As shown in Fig. 7⇓, both DC populations were extremely efficient stimulators of allogeneic splenic T cells in primary 72-h MLR and induced almost identical degrees of proliferation. The CD8α⁺ and CD8α⁻ DC were ∼10-fold more efficient compared with bulk splenocytes of the same allogeneic (B10) strain at equivalent ratios of stimulators and responders.

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FIGURE 7.

Allostimulatory capacity for naive splenic T cells (C3H) of gamma-irradiated overnight-cultured CD8α⁺ or CD8α⁻ DC isolated from B10 liver following 10 days of Flt3L administration. The data show the efficient and equivalent stimulatory capacity of sorted CD8α⁺ (▾) and CD8α⁻ (▿) DC on a per cell basis compared with that of bulk splenocytes of the same allogeneic strain (○; B10) and with that of C3H splenocytes syngeneic (•) with the responder T cells. The results were obtained from 72-h MLR and are the mean ± 1 SD from triplicate cultures. The data are representative of three separate experiments.

Trafficking of sorted liver CD8α⁺ DC to lymphoid organs of allogeneic recipients

To examine the ability of CD8α⁺ DC to migrate from the periphery to host lymphoid tissue, sorted, overnight-cultured, CD8α⁺ CD11c⁺ cells were injected s.c. into one hind footpad of normal allogeneic (C3H) mice. Two days after injection, animals were killed, and the donor CD8α⁺ DC were detected in samples of draining popliteal lymph nodes, spleen, and thymus by both mAb staining and genomic PCR for donor-specific MHC class II (IA^b). Association of donor DC with T cell areas was assessed by double immunostaining of tissue sections for donor MHC class II in combination with anti-CD3 mAb. On day 2 after their s.c. administration, most donor CD8α⁺ DC were detected in the subcapsular and paracortical sinuses of popliteal lymph nodes (Fig. 8⇓A), with very few CD8α⁺ DC in the T cell areas of the paracortex. In the spleen, CD8α⁺ DC were located in the periarteriolar lymphatic sheaths (PALS; T cell area), with most in close proximity to the arterioles (Fig. 8⇓B). In the thymus, rare donor CD8α⁺ DC were identified at the cortico-medullary junction (not shown). Donor-specific MHC class II (IA^b) DNA was detected by genomic PCR in the draining popliteal lymph node (Fig. 8⇓C), confirming migration of sorted, overnight-cultured CD8α⁺ DC from the periphery.

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FIGURE 8.

Tissue “homing” of sorted, overnight-cultured B10 (H2^b) liver CD8α⁺ DC to allogeneic C3H (H2^k) recipient draining popliteal lymph node (A) and spleen (B) 2 days after s.c. administration into the hind footpad. Trafficking of CD8α⁺ DC to T cell areas of recipient lymphoid organs was analyzed by two-color immunohistochemistry, using anti-IA^b mAb followed by ABC-AP (blue), and anti-CD3ε mAb followed by ABC-Px (red) as described in Materials and Methods. On day 2, CD8α⁺ DC (blue; arrows and inset) were located in the lymph nodes, mainly in the subcapsular and paracapsular areas. Only a few of the CD8α⁺ DC were identified in the paracortical T cell areas (red). In the spleen, CD8α⁺ DC (blue; arrows and inset) were identified in the PALS (red), and included those in close proximity to arterioles (original magnification, ×100). C, PCR detection of donor-specific DNA (MHC class II; IA^b) in the draining popliteal lymph node (LN). Amplification of β-actin was used as the internal positive control.

Comparative allostimulatory activity of sorted CD8α⁺ and CD8α⁻ DC in vivo

Next, the ability of sorted, overnight-cultured CD8α⁺ or CD8α⁻ DC to prime allogeneic T cells in vivo was assessed. Bulk lymph node cells (combined popliteal and inguinal) from C3H mice injected s.c. in the flank of one hindlimb with either CD8α⁺ or CD8α⁻ DC and killed 7 days later were used as responders in one-way, 3-day MLRs. To a fixed number of C3H lymph node T cells, variable numbers of gamma-irradiated, normal allogeneic (B10) stimulator splenocytes were added, and the proliferative response was determined. As shown in Fig. 9⇓, CD8α⁺ and CD8α⁻ liver DC were equally efficient at priming allogeneic T cells in vivo. The ex vivo proliferative responses of lymph node cells from DC-injected mice to B10 alloantigens was 5-fold greater than those of normal mouse lymph node cells due presumably to prior in vivo stimulation by either CD8α⁺ or CD8α⁻ DC.

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FIGURE 9.

Three-day MLR responses of bulk, popliteal, and inguinal (combined) lymph node cells from C3H recipients of sorted, overnight-cultured B10 CD8α⁺ (▾) or CD8α⁻ (▿) liver DC, 7 days after s.c. administration into the flank of one hindlimb. Stimulator cells were bulk splenocytes of the same allogeneic strain (○; B10), or splenocytes syngeneic with the recipient (•; C3H). The results are the mean ± 1 SD from triplicate cultures, and the data are representative of two separate experiments.