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International Immunology Advance Access originally published online on March 28, 2006
International Immunology 2006 18(5):719-728; doi:10.1093/intimm/dxl009
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Activation of thymic T cells by MHC alloantigen requires syngeneic, activated CD4+ T cells and B cells as APC

Tara M Strutt1,3,, Jude Uzonna2, Karl K McKinstry1,3, and Peter A Bretscher1

1 Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada
2 Department of Immunology, University of Manitoba, Winnipeg, Manitoba, R3E OW3, Canada
3 Present address: Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983, USA

Correspondence to: P. Bretscher; E-mail: bretschr{at}duke.usask.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We examine here the in vitro requirements to activate immunocompetent T cells, present among thymocytes, to give rise to CTL, CD4+ T cells producing IL-2 and CD8+ T cells producing IFN-{gamma}. These thymocytes are naive in not having received antigen-dependent signals characteristic of the periphery. Their activation, upon stimulation with allogeneic spleen cells depleted of T cells, referred to here as allogeneic antigen-presenting cells (APCs), to produce allo-MHC-specific effector T cells, requires activated (radiation resistant) CD4+ T cells, syngeneic with the responding thymocytes. We refer here to these T cells as ‘help’. Furthermore, optimal T cell activation requires an Ig+ B220+ cell in the allogeneic APC population, most probably a B cell. The allogeneic APCs cannot be replaced by conventional bone marrow (BM)-derived dendritic cells (DCs) activated by CD40 ligation or exposure to LPS. The requirements for both help and allogeneic B cells in the activation of thymocytes contrast with the requirements to generate substantial responses from splenic T cell populations. Activated, BM-derived DCs stimulate substantial splenic responses without help. These different requirements for activation could reflect the fact that thymocytes have not received an exit-thymus signal and/or that splenic T cells are heterogeneous, containing naive, memory and partially-activated T cells.

Keywords: B cells, cytotoxic T cells, thymocyte activation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
A valid determination of the requirements for the activation of naive T cells requires one to employ a highly purified population of antigen-inexperienced T cells. While the use of activation markers, such as CD44, CD45RB and CD62L, are useful for the identification and separation of recently activated cells from resting T cells (1–3), there is now some uncertainty as to whether a population of resting T cells characterized by CD44low, CD45RBhi and CD62Lhi expression constitutes a population of naive T cells uncontaminated with transitional or resting memory T cells (4, 5). Impurity in responding T cell populations may lead to misleading observations on the requirements to activate naive T cells. A number of studies demonstrate that thymocytes contain T cells that can be activated to generate effector T cells (6–12). We therefore decided in this study to employ thymocytes as a source of naive T cells that have not received signals characteristic of the periphery, leaving open the question for future analysis whether such T cells in the thymus have the same activation requirements as naive T cells in the periphery.

Substantial and compelling evidence in the literature shows that dendritic cells (DCs) are potent stimulators of T cell proliferation (13, 14), but the evidence that they are the only antigen-presenting cell (APC) required for the full activation of naive T cells is less clear. Some have suggested from observations obtained in anti-IgM-treated mice that B cells play an essential antigen-presenting role in the generation of adaptive immune responses (15–19). However, an assessment of the role of B cells for the activation of naive T cells in the two most widely used contemporary murine models of in vivo B cell deficiency, the µMT and the JhD B cell knockout mouse strains, have led to contradictory conclusions (20–29).

We examine here the role of different cell types required to activate naive T cells present among thymocytes when such cells are exposed to MHC-disparate cells, with special emphasis on the potential role of different APCs. Our thymocyte studies were based upon an experimental system previously established by L. M. Pilarski (8). She showed that CBA/J thymocytes are capable of generating alloantigen-specific cytotoxic T cell responses upon stimulation with allogeneic spleen cells so long as CBA/J CD4+ T cell ‘help’ is provided in the form of syngeneic-irradiated spleen cells. Using MHC compatible but Thy1 disparate CBA thymocytes and AKR spleen cells as a source of irradiated help, she demonstrated that the responding cells in culture were derived from the CBA thymocytes. A major question arising from such observations is the nature of the irradiation-resistant cell required for thymocyte responses to take place. L. M. Pilarski, employing parent into F1 radiation chimeras, provided strong evidence that the radiation-resistant CD4+ T cell is tolerant toward self-MHC antigens and displays allo-MHC specificity in that CBA/J spleen cells rendered tolerant of BALB/c antigens through the establishment of radiation chimeras could no longer help CBA/J thymocytes in the generation of anti-BALB/c alloresponses, but retained the ability to help in the generation of responses against third party alloantigens (8, 30). Employing a similar thymocyte system, we addressed whether allogeneic DCs are capable of stimulating the generation of responses from mature CBA/J thymocytes when sufficient help, in the form of irradiated syngeneic spleen, is provided, or whether the presence of additional APCs, such as B cells, are required.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
CBA/J (H-2k), C57BL/6 (H-2b) or BALB/c (H-2d) mice were obtained either from the animal colony of the College of Medicine, University of Saskatchewan (Canada) or were obtained from Charles River (Montreal, Quebec). Mice were housed under specific pathogen-free conditions. Routine screening ensured that mice were free of subclinical viral and bacterial infections. The mice employed within each experiment were of the same sex and were typically 5–8 weeks of age. Thymuses were harvested from mice not >6 weeks of age. All experiments were performed under the guidelines of the Canadian Council on Animal Care.

Antibodies
Hybridomas producing mAbs were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) or were generous gifts: TIB-207 (GK1.5) anti-CD4, TIB-99 (HO-13-4) anti-Thy1.2, TIB-211 (3.155) anti-CD8, TIB-235 (IM7.8.1) anti-CD44, TIB 146 (RA3-3A/6.1) anti-B220, HB-253 (GL1) anti-CD86, HB-301 anti-B7.1 (16-10A1) and HB-290 anti-Dec-205 (NLDC-145) were obtained from ATCC; MEL-14 (MEL14.D54) anti-CD62L and MB23G2 anti-CD45RB were gifts from I. Weissman and E. Pure, respectively. mAb was prepared from hybridoma supernatants as described (31).

T cell priming in vitro
CBA/J thymocytes in complete RPMI media (RPMI 1640 supplemented with L-glutamine [GIBCO Laboratories, Grand Island, NY, USA] containing 10% fetal bovine serum, penicillin–streptomycin [100 U ml–1] and 5 x 10–5 M ß-mercaptoethanol) were plated at 2 x 106 cells per well in 24-well costar trays (Corning, Cambridge, MA, USA). Thymocytes were stimulated with 2 x 106 cells per well BALB/c or C57BL/6 spleen cells per well, 1x 104 to 2 x 104 DCs cells per well and/or 106 B220+ B cells per well that received 1500 rads of {gamma} irradiation from a Co60 source. The number of DCs and/or B220+ cells used to stimulate thymocytes was derived from the number normally found in 2 x 106 BALB/c spleen cells (31). Irradiated CBA/J spleen cells that also received 1500 rads of {gamma} irradiation from a Co60 source were added to indicated cultures at a number 107 cells per well. Cultures were maintained at 37°C and 5% CO2 for 5–7 days. In experiments where CBA/J spleen cells were also used as responders, 2 x 106 cells per well were stimulated with alloantigen as above. Control cultures of irradiated CBA/J spleen cells stimulated with irradiated BALB/c or C57BL/6 stimulators were routinely performed. CTLs and cytokine responses were repeatedly non-existent in these cultures. All experiments presented were performed a minimum of three times.

The generation of CTLs and cytokine-producing cells was determined using the standard 51Cr CTL assay (31) employing P815 (H-2d) or EL-4 (H-2b) target cells (ATCC) and the enzyme-linked immunospot (ELISPOT) assay (32), respectively. In order to compare the generation of responses per a constant number of input thymocytes, CTLs and cytokine-producing cells were normalized to 5 x 105 input thymocytes for CTLs and 107 input thymocytes or 106 input splenocytes for cytokine-producing cells. Based on cell recoveries for responsive thymocyte cultures stimulated with allogeneic spleen cells in the presence of irradiated syngeneic spleen cells, 5 x 105 input thymocytes or 5% of culture coincides to an approximate E:T (effector to target) ratio of 10:1. Prior to seeding of effector cells in the ELISPOT assay, non-viable cells were removed by density gradient centrifugation on Ficoll® 400 (Pharmacia Biotech AB, Uppsala, Sweden).

For all experiments presented, T cells were depleted from allogeneic spleen by complement (C')-mediated lysis using Low-Tox-M-Rabbit Complement (Cedarlane, Hornby, Ontario, Canada) as described (31). T cell subsets were similarly depleted from effector cells or irradiated syngeneic spleen cell populations. All depletions were confirmed by flow cytometry as described below.

Preparation of APCs
DCs were generated from bone marrow (BM) cells as described in a protocol based on one previously established (31, 33, 34). Briefly, BALB/c BM cells were cultured in the presence of 800 Units recombinant mouse granulocyte macrophage colony-stimulating factor (GM-CSF) (BD PharMingen, Mississauga, Ontario, Canada) for 7–9 days. Every second day, BM cultures were washed and replenished with fresh media containing recombinant mouse GM-CSF. For some experiments, mature DCs were activated, prior to the addition to thymocyte cultures, by a 24-h culture period in the presence of 2.5 µg ml–1 anti-CD40 mAb (HM40-3, BD PharMingen) or 1 µg ml–1 LPS (Sigma–Aldrich, St Louis, MO, USA).

Purity and expression of co-stimulatory molecules on DCs and purity of MACS-sorted populations, discussed below, were assessed by flow cytometry using a Beckman Coulter Epics Flow Cytometer and Expo32v1.2 analysis software (Beckman Coulter, Mississauga, Ontario, Canada). {alpha}ß-Bearing T cells, CD45R (B220), MHC class II, Ig-bearing and CD19-positive B cells were detected with FITC-conjugated anti-{alpha}ß TCR, anti-CD45R, anti-MHC class II, anti-Ig or PE-conjugated anti-CD19 (Cedarlane), respectively. B7.2, B7.1, CD40 and CD205 (DEC-205) were detected by indirect staining with FITC-conjugated goat anti-rabbit IgG (H + L) or goat anti-hamster IgG (H + L) (Cedarlane). CD11c was detected with FITC-conjugated anti-CD11c antibody (BD PharMingen).

B220+ or B220– populations and Ig+ or Ig– populations tagged with rabbit anti-mouse poly-specific Ig (Cedarlane) were isolated from BALB/c spleen cells by MACS as per manufacturer's instructions, with CD45R (B220) or anti-rabbit IgG-labeled Microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). Purity was assessed by flow cytometry and was found to be >95%. Ig+ cells were removed from B220+ MACS-sorted cells by standard panning using rabbit anti-mouse poly-specific Ig (Cedarlane) (31). To ensure that B220– or Ig– MACS-sorted cells harbor splenic DCs, sorted cells were cultured overnight with 1 ng ml–1 recombinant mouse GM-CSF. Cells at a frequency and morphology of DCs that express CD11c and MHC class II were present.

Proliferation of single thymocyte cells
Proliferation of thymocytes stimulated with irradiated, allogeneic APCs in the absence or presence of syngeneic, irradiated spleen was assessed using 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). A total of 5 x 107 cells ml–1 of thymocytes were re-suspended in sterile PBS, and stained as described (35) with a final concentration of 5 µM CFSE (Molecular Probes, Eugene, OR, USA). CFSE-labeled thymocyte cultures were harvested on days 2, 3 and 4, and proliferation of single cells was assessed by flow cytometry via viable cell expression of CFSE.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The generation of CTLs and cytokine-producing cells from CBA/J thymocytes depends upon the presence of irradiation-resistant CD4+ T cells
We first set out to re-establish and explore the generality of previous in vitro findings with the thymocyte system. Thymocytes from CBA/J (H-2k) mice were stimulated with irradiated BALB/c (H-2d) spleen cells, in the absence or presence of irradiated CBA/J (H-2k) spleen cells. The generation of alloantigen-specific CTLs was then assessed on days 3, 4 and 5 against H-2d and H-2b targets. We found that more specific cytotoxicity against BALB/c alloantigens was generated by day 5 of culture in the presence of irradiated CBA/J spleen cells than in their absence, see Fig. 1(A). We have found that when we depleted the allogeneic BALB/c (H-2d) or alternatively C57BL/6 (H-2b) stimulators of T cells, the generation of anti-BALB/c (H-2d) or anti-C57BL/6 (H-2b) CTLs from thymocytes was reliably dependent upon the presence of irradiated CBA/J spleen cells, see Fig. 1(A) and (B). We employed stimulators depleted in this manner for all experiments presented. The observations presented in Fig. 1 confirm that the generation from thymocytes of allo-MHC-specific CTLs, which we have found to be CD8+ T cells (data not shown), depends upon the presence of syngeneic help (8). In addition to confirming the salient features of previous work, the observations in Fig. 1(C) and (D) show that allo-MHC-specific cells, CD8+ IFN-{gamma}- and CD4+ IL-2-producing cells are also generated from CBA/J thymocytes but only when irradiated syngeneic spleen cells or help is provided. Comparable CD8+ CTLs and IFN-{gamma}-producing cells are generated from a population of single-positive CD8+ thymocytes, isolated by antibody-dependent C'-mediated depletion of CD4+ thymocytes, when syngeneic help is present (data not shown). Similarly, comparable cytokine responses were generated by single-positive CD4+ thymocytes, isolated by antibody-dependent C'-mediated depletion of CD8+ thymocytes, in the presence of syngeneic help (data not shown). The population of cells generated from thymocytes and containing the IL-2-producing CD4+ T cells also possessed the potential to act as irradiation-resistant ‘helper’ cells for the generation of cytokine-producing cells (data not shown) and CTLs (30). From the observations in Fig. 1(E) and (F), it is evident that the generation of the allo-MHC-specific CTL responses is dependent upon both the number of thymocytes and the number of irradiated syngeneic splenic helper cells present in culture.


Figure 1
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  		Fig. 1. The generation of CTLs and cytokine-producing cells from CBA/J thymocytes is dependent upon the presence of irradiated CBA/J spleen cells. (A and B) A total of 2 x 106 CBA/J thymocytes per well were stimulated with either 2 x 106 irradiated, T cell-depleted BALB/c (filled square) or C57BL/6 (filled circle) spleen cells per well in the absence or presence (open and filled symbols, respectively) of 107 irradiated, helper CBA/J spleen cells per well. The generation of CTLs against BALB/c targets was assessed on days 3, 4 and 5 (A) and against BALB/c and C57BL/6 targets on day 6 (B) using the standard 51Cr CTL assay against P815 (H-2d) for anti-BALB/c responses or EL-4 (H-2b) for anti-C57BL/6 responses. Lysis of third party targets, EL-4 for anti-BALB/c responses or P815 for anti-C57BL/6 responses, was consistently <5%. Cells harvested from cultures containing only helper cells stimulated with alloantigen had undetectable cytotoxicity and are represented by the hatched symbols, which are hidden beneath other symbols. (C) IFN-{gamma}- and (D) IL-2-producing cells generated by stimulation of thymocytes, denoted as T, with BALB/c spleen cells in the absence or presence of helper cells, denoted as H, were detected by ELISPOT. Day 6 effectors were treated with antibody and C' to deplete various T cell subsets. Cells were untreated (1, 2, 7), treated with C' alone (3), with C' and anti-CD4, with anti-CD8 or with anti-Thy1.2 mAbs (4–6, respectively). Adjustments were not made to cell numbers to compensate for cell loss. The error bars represent the SD of antigen-dependent spots. (E) The number of input thymocytes with (filled inverted triangle, filled circle, filled triangle, filled square) or without (open inverted triangle, open circle, open triangle, open square) a constant number of 107 helper CBA/J spleen cells or (F) the number of helper CBA/J spleen cells for a constant 2 x 106 responder thymocytes per well was varied as indicated. The generation of CTLs upon stimulation with BALB/c spleen cells depleted of T cells was assessed on day 6. Some symbols representing thymocytes stimulated without help are hidden beneath others. In order to compare the generation of responses per a constant number of input thymocytes, CTLs and cytokine-producing cells were normalized to 5 x 105 input thymocytes for CTLs and 107 input thymocytes for cytokine-producing cells. Based on cell recoveries for responsive thymocyte cultures stimulated with allogeneic spleen cells in the presence of irradiated syngeneic spleen cells, 5 x 105 input thymocytes or 5% of culture coincides to an approximate E:T ratio of 10:1. Results presented are from one representative experiment of three independent experiments. Rx: treatment.

 
In order to further define the surface phenotype of the splenic helper cells, we depleted different cell subsets from the CBA/J spleen before irradiation and culture. We confirmed that the cell required is a CD4+CD8– T cell. Further observations, which are presented in Table 1, show that the ‘helper activity’, required to support thymocyte responses, can be abrogated by the removal of CD44hi cells, but not by the removal of cells bearing high levels of CD45RB or CD62L. This surface phenotype of CD44hi, CD45RBlow and CD62Llow is the same as ‘naturally’-activated CD4+ T cells found in normal mice (36, 37). Collectively, these observations lead us to suggest that the syngeneic (radiation resistant) CD4+ Th, which are required for the generation of allo-specific thymocyte responses and are present in spleen, are activated T cells.


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  		Table 1. Characterization of the CD4+ T cells present in irradiated CBA/J spleen that help thymocytes

 
Allo-MHC-bearing DCs derived from BM support the generation of CTL responses by CBA/J spleen cells, but do not support the generation of CTL responses by CBA/J thymocytes
We wished to address the question of which APC in the allogeneic, T cell-depleted spleen cell population is required to stimulate the generation of ‘helper-dependent’ CTLs and cytokine responses by thymocytes. In order to determine if DCs can stimulate the generation of responses by thymocytes, we chose to employ conventional BM-derived DCs as allogeneic stimulators.

To confirm that the cells generated from BM progenitor cells in vitro are conventional DCs, we examined the ability of differentially matured, BM-derived, BALB/c DCs to support the generation of CTLs and cytokine responses from CBA/J spleen cells. Before irradiation and culture, the DCs were matured for 24 h either in the absence of exogenous stimuli, or were activated by ligation of CD40 or by exposure to LPS. The observations in the right panel of Fig. 2(A) show that 104 activated DCs could stimulate the generation of alloresponses from un-irradiated, CBA/J splenic responder cells, with LPS-activated DCs stimulating the most vigorous responses. However, the same DCs fail to stimulate the generation of responses from CBA/J thymocytes, see the left panel of Fig. 2(A), even when irradiated syngeneic CBA/J splenic helper cells are provided. The ability of the BM-derived DCs to stimulate the generation of alloresponses from splenocytes supports the position that the cells generated are in fact conventional DCs. This inference is further supported by the observation that the BM-derived cells up-regulate upon exposure to LPS the standard co-stimulatory molecules, B7.1, B7.2 and CD40 more so than the DC marker CD205 (DEC-205), see Fig. 2(B). We have also confirmed that the BM-derived DCs matured by exposure to LPS for 24 h dramatically down-regulate their ability to present ovalbumin (OVA) to OVA-specific BALB/c T cell clones derived from mice primed with OVA in CFA (data not shown), as assessed by OVA-dependent T cell proliferation.


Figure 2
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  		Fig. 2. Allo-MHC-bearing DCs derived from BM support the generation of responses from spleen, but fail to support the generation of CTL responses from thymocyte responders. (A) CBA/J thymocytes, 2 x 106 cells per well, were stimulated with 2 x 106 irradiated, T cell-depleted BALB/c spleen cells per well in the absence or presence of 107 irradiated CBA/J spleen cells per well (open square or filled square, respectively). Separate CBA/J thymocyte cultures of 2 x 106 cells per well that contained irradiated syngeneic help or 2 x 106 CBA/J splenocyte responders per well were also stimulated with 104 BALB/c BM-derived DCs per well. Prior to the addition to cultures, BM-derived DCs were matured for 24 h in the presence of complete media (filled circle), anti-CD40 mAb (filled triangle) or 1µg ml–1 LPS (filled inverted triangle). CTL responses were assessed against labeled P815 (H-2d) target cells or against labeled third party targets, EL-4 (H-2b). CTL responses against H-2d targets is shown; lysis of third party H-2b target cells is shown in the inset. Some symbols are hidden beneath others, reflecting specific cytotoxicity of <5%. (B) The expression of B7.2, B7.1, CD40, CD205, B220 or {alpha}ß-TCR on BM-derived cells was assessed by flow cytometry. Background staining is represented as shaded histograms. Expression profiles after maturation in the presence of complete media (thin curve) or LPS (thick curve) is shown. Similar observations were obtained in three independent experiments.

 
While effector T cells fail to be generated when thymocytes are stimulated with LPS-activated BM-derived DCs, even when irradiated, CBA/J spleen cell help is provided, see left panel of Fig. 2(A), we have found that allogeneic DCs, in the presence of irradiated CBA/J CD4+ T cells, can stimulate thymocytes to divide. Thymocyte division is evident from the loss of CFSE intensity over time, see Fig. 3. The bulk of this division appears to occur between days 2 and 4 of culture. Whether the proliferating cell is a single-positive CD4, CD8 T cell or possibly even a double-positive thymocyte warrants further investigation. We are currently exploring this issue as well as the functional capacity of such cells after different rounds of division.


Figure 3
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  		Fig. 3. Thymocytes stimulated with allogeneic BM-derived DCs in the presence of syngeneic irradiated help are stimulated to divide. CBA/J thymocytes, at 2 x 106 cells per well, were stimulated with 2 x 104 irradiated DCs per well derived from the BM of BALB/c mice or with 2 x 106 irradiated, T cell-depleted, BALB/c spleen cells per well, either in the absence or presence of 107 irradiated CBA/J spleen cells per well acting as help. Responding CBA/J thymocytes were labeled with CFSE prior to the addition to culture. Cell division of viable cells recovered from density gradient centrifugation on Ficoll hypaque was assessed at 24-h intervals on days 2 (thin curve), 3 (dotted curve) and 4 (thick curve) by the loss of CFSE fluorescence intensity. Background fluorescence of unlabeled and stimulated thymocytes is indicated by the shaded histogram. The results presented are from one experiment representative of three independent experiments.

 
Allo-MHC-bearing B220+Ig+ cells support the generation of CTLs and cytokine-producing cells from thymocytes when help is provided
The inability of mature BM-derived DCs to activate thymocytes to produce CTLs or cytokine-producing T cells led us to characterize the cell present in the T cell depleted, allogeneic spleen cell population that can stimulate thymocytes to produce such activated T cells in the presence of help. We wished to assess the potential role of B cells as APCs. Most B220+ cells are B cells (38). We therefore sorted B220+ cells from BALB/c spleen, and used the positive fraction as allogeneic stimulators for CBA/J thymocytes or for splenocytes. We also examined the capacity of the combination of BM-derived DCs and MACS-sorted B220+ cells to stimulate the generation of allogeneic responses from thymocytes. The generation of IFN-{gamma}-producing cells was slightly greater when both B220+ and BM-derived DCs were present in culture than when B220+ cells alone were employed as alloantigen, see Fig. 4. This observation leads to the possibility that the presence of both types of APCs has a cooperative effect in the generation alloantigen-specific IFN-{gamma}-producing cells. However, such cooperation was not evident for the generation of alloantigen-specific CTLs, see Fig. 4. We depleted the B220+ cells of Ig-bearing cells and assessed how such depletion affected the ability of the cells to stimulate thymocytes to produce CTLs and IFN-{gamma}-producing cells in the presence and absence of help, see Fig. 4(A) and (C), group 5. The generation of CTLs and IFN-{gamma}-producing cells was dramatically decreased but less striking findings were obtained for the generation of IL-2-producing cells from thymocytes (data not shown). The CTL responses in cultures of thymocytes stimulated with BM-derived DCs were below detection (<5% lysis) and are therefore not readily seen in Fig. 4(A). Of particular note is the lack of a detectable response stimulated by DCs and B220+ Ig– spleen cells, denoted by the + symbol. These findings lead us to suggest that the presence of Ig+ B220+ APCs, which excludes Ig– B220+ DCs, is required for the generation of primary, allo-MHC-specific T cell responses from competent naive T cells among thymocytes. These observations provide strong evidence that B cells are required to generate these responses.


Figure 4
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  		Fig. 4. Allo-MHC-bearing B220+ Ig+ spleen cells support the generation of CTLs and IFN-{gamma}-producing cells from thymocytes. (A) CBA/J thymocytes, 2 x 106 cells per well, were stimulated with combinations of irradiated BALB/c APCs in the absence or presence of 107 irradiated CBA/J spleen cells per well (open and filled symbols, respectively). DCs were derived from BM, and B220+ cells isolated from the spleens of BALB/c mice using MACS beads. Thymocytes were stimulated with 104 DCs per well (filled square), 104 DCs plus 106 B220+ cells per well (filled circle), 104 DCs plus 0.5 x 106 B220+ cells per well (filled triangle), 104 DCs plus 106 B220+ cells per well depleted of Ig+ cells (+) or with 106 Ig+ B220+ cells per well (filled diamond). Many CTL responses were <5% specific lysis and are not readily evident in the figure. (B) CBA/J spleen cells, 2 x 106 cells per well, were also stimulated with either 104 DCs per well (filled square) or 106 Ig+ B220+ cells per well (filled diamond). The ability of the different APCs to support the generation of CTLs was determined on day 7 against labeled P815 (H-2d). The corresponding IFN-{gamma} ELISPOT in which 104 thymocyte derived effectors per well were seeded in the ELISPOT with 2.5 x 105 irradiated stimulators per well is shown in (C). The asterisk represents 104 DCs plus 0.5 x 106 Ig+ B220+ cells per well, and ± represents 104 DCs plus 106 B220+ cells per well depleted of Ig+ cells. The number of antigen-dependent (Ag Dept) spots for each condition is also shown. The errors represent the standard deviation. Similar findings were obtained in three independent experiments.

 
We also assessed the ability of B220– and/or Ig– MACS-sorted fractions of BALB/c splenocytes, depleted of T cells, which mainly contain allo-MHC-bearing DCs and macrophages for the former fraction and mainly allo-MHC-bearing DCs for the latter, to stimulate the generation of responses from immunocompetent thymocytes given irradiated help. The B220– cell fraction isolated from BALB/c spleen cells stimulates the generation of CTL responses from CBA/J spleen cells, but fails to optimally stimulate the generation of CTLs from thymocytes, see Fig. 5. Similarly, the generation of IFN-{gamma}-producing cells was markedly reduced, and the generation of IL-2-producing cells was reduced 2-fold upon stimulation with B220– spleen cells (data not shown). These observations support a role for B220+ cells as allogeneic APCs, which are likely to be B cells. However, smaller but significant responses are seen with B220– allogeneic APCs. The allogeneic APCs responsible for these responses could be B220– B cells (39). All B cells are Ig+, and so we examined the ability of Ig– allogeneic APCs to stimulate the production of specific cytokine-producing cells. When Ig– fractions isolated from BALB/c spleen were employed to stimulate the generation of responses from CBA/J spleen cells and thymocytes, see Fig. 5(B), only splenocytes generated substantial IFN-{gamma}- and IL-2-producing cells. The B220+ or Ig+ fractions, which operationally are primarily B cells, stimulated the generation of responses from both CBA/J thymocytes and splenocytes in a manner similar to that generated upon stimulation with irradiated, T cell-depleted BALB/c spleen. Flow cytometry analysis of the percentage of CD4+- and CD8+-bearing cells present in such cultures on day 6 revealed no significant difference in the effector populations generated from thymocytes in the presence of help when T cell-depleted spleen or Ig+ fractions of this population are used as stimulators (Fig. 6, panels D and E, respectively). Similarly, the CD4+ and CD8+ T cells generated from spleen responders are very similar when stimulated by the same APC populations (Fig. 6, panel G and H). This indirectly implies that the antigen-dependent generation of these cells is due predominantly to an Ig+ cell in the T cell-depleted spleen.


Figure 5
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  		Fig. 5. Allo-MHC-bearing B220– or Ig– spleen cells fail to support the generation of optimal T cell responses from thymocytes, but do support the generation of responses from splenocytes. CBA/J thymocytes, denoted as T, at 2 x 106 cells per well, or a similar number of splenocytes, denoted as S, were stimulated with different irradiated BALB/c APCs. Thymocytes were stimulated in the absence or presence of 107 irradiated CBA/J spleen cells per well (open and filled symbols, respectively). B220+ and B220– populations for (A) or Ig+ and Ig– populations for (B) were isolated from the spleens of BALB/c mice using MACS beads. Responding thymocytes or splenocytes were stimulated with 2 x 106 anti-Thy1.2-treated spleen cells per well (filled square), 106 B220+ cells per well (filled triangle) or anti-Thy1.2-treated B220– cells derived from 2 x 106 cells (filled circle). The ability of the different APCs to support the generation of CTLs (A) was determined on day 7 against labeled P815 (H-2d) and EL-4 (H-2b) target cells. Specific lysis of third party EL-4 target cells by effector cells generated by stimulation with anti-Thy1.2-treated spleen is represented by the hatched square. The ability of the different APCs to support the generation of cytokine-producing cells was determined in the ELISPOT assay (B). The asterisk represents anti-Thy1.2-treated Ig– cells derived from 4 x 106 BALB/c spleen cells. Similar findings were obtained in three independent experiments.

 

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  		Fig. 6. Similar CD4+ and CD8+ percentages in cultures of CBA/J thymocytes or splenocytes after stimulation with irradiated anti-Thy1.2-treated spleen or Ig+ BALB/c alloantigens. CBA/J thymocytes were stimulated in the absence or presence of 107 irradiated CBA/J spleen cells per well (Irrad Spleen) as described in the legend of Fig. 1. CBA/J thymocytes or splenocytes were stimulated with 2 x 106 anti-Thy1.2-treated spleen cells per well (Spl), 106 anti-Thy1.2-treated Ig+ BALB/c spleen cells per well or anti-Thy1.2-treated Ig– cells derived from 2 x 106 cells. The percentage of CD4+ or CD8+ present on day 6 is shown. The percentage of CD4+ and CD8+ cells present in day 0 CBA/J splenocytes was found to be 14.3 and 7.7%, respectively. Similar profiles were observed in three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The observations reported here appear to demonstrate that the optimal activation of thymocytes, or possibly even any significant activation to produce fully functional effector T cells, requires B cells as APC as well as activated Th. We find that various sources of DCs are either not effective or not as effective as Ig+ cells, which are presumably B cells, in their role as APC. Moreover, these requirements for activation do not apply to the responding T cells present in spleen cell populations.

These observations lead to a number of questions. The overriding question is whether the requirement for activated CD4+ T cells and B cells as APC in the activation of thymocytes represents what is required to activate naive T cells in general, or whether these requirements are particular to thymocytes as they have not received a signal on exiting the thymus (40). We have not addressed this question here, but it may be possible to employ peripheral TCR transgenic CD4+ cells from RAG–/– mice as a relatively pure source of naive T cells to study the requirements to activate antigen-inexperienced cells. This T cell population is expected to have no or fewer activated T cells than the corresponding population of TCR transgenic cells obtained from mice with a wild-type background.

We are aware that our in vitro observations must be interpreted with caution when reflecting on what occurs in vivo. In particular, observations have led to the suggestion that naive T cells stimulated by antigen-bearing DCs differentiate and migrate to sites where they can interact with B cells (41–43). Thus, DCs may have important in vivo functions not readily apparent from in vitro studies.

The evidence for a central role for B cells in the activation of naive T cells is contradictory (15, 17, 20, 25, 44). Observations from several studies employing µMT B cell-deficient mice have led to the conclusion that B cells are not required for the induction of cellular and humoral responses. However, it is now apparent that µMT mice, at least on the BALB/c background, harbor some B cells (45, 46). Other studies, employing a less ‘leaky’ model of B cell deficiency, JhD B cell knockout mice, have led to the conclusion that B cells are essential for the activation of T cell responses (23, 25). These studies seem to suggest that the involvement of B cells in T cell activation reflects a mechanism other than the production of secreted antibody (25). It has been argued that observations supporting such a role for B cells could be explained by a requirement for B cells in the development of proper lymphoid architecture (47); our in vitro findings favor the possibility that B cells are, in addition, essential APCs involved in delivering the required cytokines and/or co-stimulatory signals to naive T cells undergoing activation. The ability of DCs to act as potent stimulators for ‘naive’ T cells in a number of studies (14, 48–52) may be explicable by the presence of resting, memory or partially-activated T cells that are ‘masquerading’ as naive T cells (53). Again, the use of TCR transgenic mice in a RAG–/– background might help to resolve whether naive CD4+ T cells can really be fully activated by DCs. Peripheral T cells from such mice are anticipated to be in a naive state, as they do not contain bi-specific TCR that arise because of endogenous TCR{alpha}, and potentially also TCRß expression that might result in their activation by diverse antigens (54–56).

Lastly, it is natural to wonder whether our findings are pertinent to the in vivo requirements to activate naive T cells by non-MHC antigens. We consider the relevance of these findings to such activation of naive T cells must be assessed experimentally. We are currently attempting to develop an in vivo system to assess the role of activated T cells and B cells in supporting the activation of thymocytes and naive T cells by non-MHC antigens. The requirement for activated CD4+ T helper cells for the generation of primary immune responses, if general, leads to the question of the origin of the first activated CD4+ T cells. It is known that the in vivo requirements for the activation of murine CD4+ T cells is less stringent in the early stages of the development of the immune system than in a fully developed animal (57, 58). It is conceivable that this phenomenon, which is due to the lymphopenic environment of the neonate, could be related to how the first activated or natural memory CD4+ T helper cells arise.

Our findings naturally lead to the question of what happens to naive thymocytes when they are exposed to antigen on DCs, without interaction with B cells and the activated radiation-resistant CD4+ T cells required for their activation. Allogeneic DCs isolated from the spleen can specifically abrogate the responsiveness of at least thymocyte CD8+ T cells when they interact in fetal thymic organ cultures derived from day 14 embryos (59). This observation would mean, if mature DCs were the only APCs required to activate naive T cells, that thymocytes are intrinsically only inactivatable on interacting with antigen. This interpretation has been favored in the past (59). Our observations suggest an alternative possibility, namely that the lack of B cells and/or activated CD4+ T cells may not allow for full activation to take place, and instead, interaction with antigen under these circumstances might lead to T cell inactivation. The requirement for B cells and activated CD4+ T cells in the activation of naive CD4+ T cells may be important in ensuring, under most circumstances, that CD4+ T cells specific for peripheral self-antigens are not activated (60). Most of our findings involve the requirement to generate CTLs and IFN-{gamma}-producing CD8+ T cells. The requirements for generating IL-2-producing CD4+ T cells seem similar. We also examined whether substantial numbers of IL-4-producing CD4+ T cells are generated in this system. The marginal response seen prevented us from analyzing the requirements to generate such T cells.


   Acknowledgements
 
We thank H. Bull and V. Misra, and Duane Hamilton for their review of the manuscript. This work was supported by a Canadian Institute for Health Research grant MOP 14121. T.M.S. was supported by a Natural Sciences and Engineering Council of Canada Post-Graduate Award.


   Abbreviations
 
APC, antigen-presenting cell
ATCC, American Type Culture Collection
BM, bone marrow
C', complement
CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester
DC, dendritic cell
ELISPOT, enzyme-linked immunospot
GM-CSF, granulocyte macrophage colony-stimulating factor
OVA, ovalbumin

   Notes
 
Transmitting editor: A. Singer

Received 2 August 2005, accepted 14 February 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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