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International Immunology Advance Access originally published online on September 7, 2009
International Immunology 2009 21(11):1213-1224; doi:10.1093/intimm/dxp085
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© The Japanese Society for Immunology. 2009. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD4 T cell cooperation is required for the in vivo activation of CD4 T cells

Nathan C. Peters1,2, David R. Kroeger1, Steven Mickelwright1 and Peter A. Bretscher1

1 Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5
2 Present address: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Building 4, Room B1-12, 4 Center Drive, Bethesda, MD 20892-0425, USA

Correspondence to: P. A. Bretscher; E-mail: peter.bretscher{at}usask.ca or N. C. Peters; E-mail: NPeters{at}niaid.nih.gov


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
We address here the role of CD4 T cell cooperation in the activation of CD4 T cells. Administration of aggregated hen egg lysozyme (HEL) without microbial adjuvant to BALB/c mice normally generates cytokine-producing CD4 T cells specific for the HEL major peptide, HEL105–120, as well as CD4 T cells specific for HEL non-major peptides. The prior administration of HEL105–120 ablates the generation of cytokine-secreting CD4 T cells specific for HEL105–120, as well as the CD4 T cells specific for HEL non-major peptides, normally generated upon HEL challenge. Thus, the activation of HEL non-major peptide-specific CD4 T cells appears to depend upon the HEL105–120-specific CD4 T cell population. In contrast, when HEL105–120 and saline-treated mice are challenged with HEL coupled to ovalbumin (OVA), CD4 T cell responses to HEL non-major peptides and to OVA are the same, whereas treated mice still do not generate cytokine-secreting cells specific for HEL105–120. We infer that the administration of HEL105–120 does not generate regulatory cells capable of down-regulating CD4 T cell responses to HEL and OVA peptides. OVA-specific CD4 T cells restore the generation of HEL non-major peptide-specific T cells in the absence of HEL major peptide-specific T cells. We conclude that the generation of CD4 T cells producing IL-2, IFN-{gamma} and IL-4 requires CD4 T cell cooperation and that this cooperation is not mediated simply by CD40–CD40L interactions. We also conclude from these observations that there is no requirement for a microbial or danger signal for CD4 T cell activation.

Keywords: CD4 T cell activation, CD4 T cell cooperation, peripheral tolerance


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
The antigen-dependent, primary activation of most B cells to produce antibody (1–4), and of at least some CD8 T cells to produce CTL or memory CTL's (5–9), requires activated CD4 T cells. Antigen can ‘inactivate’ these B and CD8 T cells in the absence of CD4 T cell-mediated help, rendering the B cells (10, 11) and CD8 T cells (7, 12) unable to respond to subsequent activation signals. Thus, CD4 T cells play a central role in both the fate of other lymphocytes and in controlling the initiation of diverse immune responses.

Observations show that the activation of naive CD4 T cells involves a two-signal mechanism. Activation requires a TCR-mediated signal, signal 1, generated upon TCR recognition of peptide in the context of MHC class II molecules as well as a second signal, signal 2, generated when co-stimulatory (CoS) molecules on antigen-presenting cells (APC) interact with counter receptors on the CD4 T cell (13–15). It is also recognized that the generation of signal 1, without the generation of signal 2, results in the inactivation of the CD4 T cell, rendering it unresponsive to subsequent activation signals (12–14).

Two models for what determines whether antigen activates or inactivates naive CD4 T cells are cast within the context of this two-signal framework for the activation of CD4 T cells. The models differ in their postulates as to what controls the generation of the critical signal 2 required for activation but not for inactivation. According to Microbial and Danger Signal Models, the expression of CoS molecules by APC requires APC activation via a ‘microbial/danger’ signal generated when certain APC receptors, most often toll-like receptors (TLRs), bind molecular patterns ubiquitously present in microorganisms (16–18) or cellular products that arise upon exposure to danger (19, 20). Whether antigen activates a CD4 T cell depends, according to these Microbial/Danger Signal Models, only on the circumstances at the time of immunization. In contrast, a version of the original Two-Signal Model (21, 22), consonant with contemporary observations (23), states that the expression by the APC of the critical CoS molecules normally requires activation of the APC by other antigen-specific CD4 T cells and hence CD4 T cell activation requires CD4 T cell cooperation (21–24). Recent observations show that the occurrence of CD4 T cell-dependent responses, and their enhancement by classical adjuvants such as CFA, does not depend upon TLR-mediated signaling (25). This study leads to the suggestion that another mechanism of APC activation may be required for the activation of CD4 T cells, possibly mediated by CD4 T cell cooperation.

Most CD4 T cells specific for self-antigens present in the thymus are either deleted or rendered anergic as they are generated (23, 26). It is clear, however, that T cells specific for self-antigens, not present at sufficient levels in the thymus, emigrate and have the potential for causing organ-specific autoimmunity (27, 28). There is a need for the control of the activation of such CD4 T cells if organ-specific autoimmunity is to be avoided. One means to achieve this may be via the thymus-dependent generation of self-antigen-specific T regulatory cells that control the activation of organ-specific cells in the periphery (28, 29). Another potential means is via a mechanism of CD4 T cell activation that requires CD4 T cell cooperation, and where the interaction of a single CD4 T cell with antigen results in its inactivation (21–23). In this case, a peripheral self-antigen, first appearing early in ontogeny and being present continuously thereafter, is expected to inactivate peripheral CD4 T cells specific for this antigen as they are generated and emigrate from the thymus. In contrast, CD4 T cells specific for a foreign antigen can accumulate in its absence and can then cooperate, resulting in an immune response, when the foreign antigen impinges upon the immune system.

The primary aim of the studies reported here was to test whether CD4 T cell cooperation is involved in the process of the in vivo activation of endogenous CD4 T cells. In order to achieve this, we employed an experimental system in which we had previously defined the peptide specificity of all the cytokine-secreting CD4 T cells specific for hen egg lysozyme (HEL), following immunization with this protein administered without microbial adjuvant. The validity of this statement on the specificity of the CD4 T cells generated is shown by the fact the sum of the numbers of cytokine-producing cells specific for individual but non-overlapping HEL peptides equals the number of cytokine-producing cells specific for the whole protein (30).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Mice
BALB/c mice were obtained from the animal colony of the College of Medicine, University of Saskatchewan (Canada) or from Charles River (Montreal, Canada). Mice were of the same sex within each experiment and at the time of immunization were 6–8 weeks of age. All experiments were carried out in accordance with standards approved by the Canadian Council on Animal Care.

Preparation of HEL, HEL peptides and HEL–OVA
Soluble (sol) and heat-aggregated hen egg lysozyme (ha-HEL) were prepared as previously described (30). LPS was removed from solublized Grade VI HEL (Sigma) preparations (50 mg ml–1) by absorption with Detoxi-Gel endotoxin removing gel (Pierce) according to the manufacturer's instructions. Briefly, 10 ml of the sol-HEL preparation was passed through a column packed with Detoxi-Gel endotoxin removing gel. LPS-free sol-HEL was then heat aggregated as previously described (30). All non-disposable laboratory equipment encountering the HEL preparation was washed using E-TOXA-CLEAN (Sigma) to remove any bound LPS. The final concentration of LPS in heat-aggregated antigen, prepared from sol-HEL either untreated or decontaminated of LPS by passage over endotoxin removing gel, was determined using the semi-quantitative E-TOXATE test (Sigma), according to the manufacturer's instructions. Briefly, sol- and heat-aggregated HEL was pH adjusted before use in the E-TOXATE test with endotoxin-free NaOH and HCl (Sigma). Serial dilutions were made of an endotoxin standard supplied with the E-TOXATE test, as well as of those samples in which the concentration of LPS was to be determined. The concentration in the sample was estimated based upon the highest dilution that showed a positive result following the addition of the E-TOXATE active reagent. Due to the fact that the concentration of LPS in 1 mg of HEL passed through the Detoxi-Gel column was below the detection limit of the E-TOXATE test, we determined the amount of LPS in starting preparations containing 15–20 mg of HEL per ml–1 and thereby estimated the amount of LPS present in 1 mg of ha-HEL and subsequently in the 100 µg used for immunization. Aluminum hydroxide adjuvant (ALUM) used for immunization was found to be free of contaminating LPS.

The Alberta Peptide Institute (University of Alberta, Edmonton, Canada) or Research Genetics (Huntsville, USA) produced synthetic peptides. All peptides used in the studies reported here were routinely purified by HPLC to >95% purity before we received them. Peptides were stored for the long term in lyophilized form. Peptides were dissolved in deionized and de-gassed water, filtered and stored in the short term in 1 ml aliquots with N2 gas in the headspace. Peptide aliquots were periodically checked for bacterial contamination using blood agar plates.

The HEL–ovalbumin (OVA) ‘conjugate’ was prepared as follows. A 40 mg ml–1 solution of HEL in 0.85% saline was incubated in a 100°C water bath until the solution began to aggregate, ~10 min after incubation. Prior aggregation of HEL alone, before the addition of OVA, was required to prevent the formation of predominantly OVA–OVA conjugates as OVA aggregates at a faster rate then HEL. The partially aggregated HEL solution was allowed to cool to room temperature and an amount of a solution of OVA at 10 mg ml–1 of OVA in 0.85% saline was added to achieve a 1:1 molar ratio of the two proteins. The mixture was then heated in a 100°C water bath for 5 min, resulting in copious aggregation. The tube was allowed to reach room temperature and spun to separate the precipitate from the supernatant. The protein content of the aggregate was assessed and prepared as previously described (30).

Generation of immune responses
BALB/c mice were immunized with 7 nmol of HEL105–120 in normal saline emulsified 1:1 in CFA containing 1 mg ml–1 killed Mycobacterium tuberculosis strain H37Ra (Difco Laboratories, Detroit, MI, USA) subcutaneously in the skin at the base of the tail in a final volume of 100 µl over two injection sites. BALB/c mice were also immunized intraperitoneally (i.p.) with 100 µg (7 nmol) sol- or ha-HEL in normal saline, or in normal saline absorbed 1:1 with Alhydrogel 2% adjuvant (ALUM) (Bio-Phos, Denmark), in a total volume of 300 µl. Some BALB/c mice were immunized with 100 µg of ha-HEL–OVA in normal saline absorbed 1:1 with Alhydrogel 2% adjuvant (ALUM) (Bio-Phos), in a total volume of 300 µl. Control mice received either 100 µl of saline per CFA, 300 µl of saline or 300 µl of saline per ALUM. In some experiments, mice were pre-treated with 300 µg of HEL105–120 in saline on day 13 and 100 µg on days 6 and 3 before immunization in an attempt to render them unresponsive to this peptide. In some cases, we examined whether 100 µg of the anti-CD40 agonist mAb FGK4.5 (BioXcell, NJ, USA), given i.v., could enhance the activation of HEL peptide-specific CD4 T cells when administered along with our standard challenge of ha-HEL on ALUM. We chose a standard dose of the anti-CD40 agonist FGK4.5 that, given at the time of challenge, increased a small anti-HEL response generated by giving a low dose of ha-HEL without adjuvant (data not shown). The administration of FGK4.5 results on day 10 in a 2-fold increase in the cellularity of the spleen with no change in proportion of CD4 T cells. For these experiments, the size of the responses is reported as antigen-specific cytokine-secreting cells (CSC) per spleen.

Optimal detection of antigen-specific CSC using the ELISPOT assay
Lymph node (LN) and spleen cells were prepared for use in a modified enzyme-linked immune spot (ELISPOT) assay as described previously (31). Briefly, LN and spleen cells were plated in the ELISPOT assay at several densities with additional cells from unprimed mice to maintain the same total number of cells per well. Using this methodology, the number of antigen-dependent spots is linearly dependent on the number of sensitized cells plated (30, 31).

HEL antigen was added to APC suspensions at the optimal concentration of 100 µg (7 nmoles) of ha-HEL or 400 µg (28 nmoles) of sol-HEL per well. HEL peptides were added to APC suspensions at the optimal concentration of ≥0.7 nmoles per well (3.5 µM) unless otherwise indicated. ELISPOT-forming cells for each data point were assessed in triplicate unless otherwise indicated. The number of spots reported as antigen specific is the number of spots generated in the presence of antigen minus the number generated in the absence of antigen. Plotted data represent antigen-dependent responses in antigen+ wells, which were significantly greater than responses in antigen– wells (two-sample t-test, assuming unequal variances, P < 0.05). Two-sample t-tests, assuming unequal variance, were performed using Excel (Microsoft Corporation). All P-values reported are two sided.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Immune responses to HEL administered without microbial adjuvant
We first set out to explore whether substantial immune responses to purified HEL could be generated without microbial adjuvant. Classical studies demonstrate that aggregates of proteins are much more immunogenic than soluble protein (32). We therefore prepared heat-aggregated HEL (ha-HEL) as described (30) and found conditions where ha-HEL, in contrast to sol-HEL, generates substantial immune responses even when given without any adjuvant, see Fig. 1. Aggregation clearly increases the immunogenicity of this protein as HEL-specific IL-2- and IL-4-producing cells were detected in all mice tested 10 days following administration of 100 µg of ha-HEL in saline on days 0 and 3. In contrast, the administration of sol-HEL in saline did not generate responses significantly above those detected in naive controls. We wanted to establish whether or not this response was dependent on LPS in the antigen preparation, as the presence of this microbial product might allow a non-microbial antigen, such as HEL, to induce CD4 T cells, and commercially available HEL contains this immunomodulatory substance (33). We determined the level of LPS in our stocks of HEL as ~5 ng mg–1 of protein using a commercially available endotoxin test, and we used standard procedures to decrease the amount of LPS by at least a 100-fold, see Methods. This resulted in <0.003 ng of LPS per 100 µg of HEL injected, ~1000- to 10 000-fold lower than that amount of LPS reported to affect immune responses (33–35). The immune response generated by the LPS-depleted antigen preparation was indistinguishable from that generated by the LPS-containing starting material, see Fig. 1. We conclude that the anti-HEL response we see is likely not dependent on to contaminating LPS in our HEL. The response to ha-HEL was dominated by IL-4-producing cells, as might be expected following the administration of an exogenous, aggregated, protein antigen. While this finding is suggestive that immune responses to a simple protein are generated in the absence of LPS, the most likely microbial contaminant, it does not definitively exclude a role for microbial or danger signals involved in the activation of HEL-specific precursor Th cells, as there may be other microbial or noxious products in our antigen preparation.


Figure 1
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  		Fig. 1. Generation of immune responses to HEL administered without microbial adjuvant. Three to four female BALB/c mice per group were immunized i.p. on days 0 and 3 with saline, 100 µg of sol-HEL, heat-aggregated HEL (ha-HEL) or LPS-free heat-aggregated HEL, (LPS-free ha-HEL). HEL-specific IL-2- (circles), IFN-{gamma} (triangles) and IL-4- (squares) producing spleen cells were enumerated on day 10 post-immunization using the ELISPOT assay (28, 29). Results are representative of three to five similar experiments. IFN-{gamma}- and IL-2-producing cells from saline-immunized (n = 12) or unprimed (n = 7) control mice were not detected. IL-4-producing cells from saline-immunized or unprimed mice ranged from 0–12 per 106 spleen cells. Each data point represents the mean of triplicate wells from an individual mouse. Lines represent the mean of means ± SEs. Plotted data represent antigen-dependent responses, which were significantly greater than responses in antigen– wells (P < 0.05).

 
Activation by HEL of CD4 T cells specific for one HEL peptide operationally depends on functional CD4 T cells specific for other HEL peptides
We wished to more critically test the hypothesis that CD4 T cell cooperation is involved in the in vivo activation of CD4 T cells. Critical to our approach was a modification of the ELISPOT assay for optimally detecting single, antigen-specific, cytokine-secreting T cells (30, 35). We have shown that the efficiency of detecting HEL peptide-specific CD4 T cells is the same when an optimal amount of HEL protein or of the immunizing HEL peptide is present in the assay (30). We were able, employing this assay, to account for the specificity of essentially all the cytokine-secreting, HEL-specific CD4 T cells, generated upon immunization with the HEL protein, in terms of their reactivity to a set of non-overlapping HEL peptides in both BALB/c and CBA mice (30). The CD4 T cell response to HEL in BALB/c mice is dominated by T cells specific for HEL105–120, with about half of the responding CD4 T cells being specific for this peptide. The other CD4 T cells are specific for ‘minor’ peptides, with consistent responses to the ‘major minor’ peptide, HEL11–25 (30).

We surmised that if CD4 T cell cooperation is required to activate naive CD4 T cells specific for one peptide of an antigen and then the activation of these peptide-specific CD4 T cells should depend upon the presence of CD4 T cells specific for other peptides derived from the same nominal antigen. Some experimental evidence supports such a dependence (36–38). We therefore examined the requirement for CD4 T cell cooperation by assessing the activation of CD4 T cells specific for certain peptides of an antigen both in the presence and the absence of other, potentially cooperating CD4 T cells, specific for other peptides of the same nominal antigen.

In order to eliminate potentially cooperating CD4 T cells in an in vivo setting, we administered in saline high doses of the dominant peptide, HEL105–120, intravenously to BALB/c mice on days 13, 8, and 3 before our challenge based upon a protocol developed to inactivate peptide-specific CD4 T cells (39,40). To test the efficacy of our peptide-mediated inactivation, we challenged saline-injected normal mice (‘N’) and HEL105–120-treated and putatively tolerant mice (‘T’) with HEL105–120 in CFA on day 0, thirteen days following the first peptide-treatment, and assessed the CD4 T cell response in the spleen and LN on days 6 and 10 after immunization, see Fig. 2. Prior administration of HEL105–120 greatly decreased the peptide-specific CD4 T cells producing IL-2, IFN-{gamma} and IL-4, normally generated on challenge with this peptide administered in CFA. This operational unresponsiveness, affecting the generation of CD4 T cells producing cytokines characteristic of both Th1 and Th2 responses and reflecting responses in different lymphoid organs, is most likely due to the virtually complete elimination of CD4 T cells specific for the major peptide and is unlikely to be due to the generation of antigen-specific suppressor T cells (39–41). Administration of a peptide resulting in the inactivation of CD4 T cells specific for that peptide does not affect the subsequent activation of CD4 T cells specific for a second, unrelated, peptide following challenge with this second peptide in CFA, showing inactivation is peptide specific (data not shown).


Figure 2
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  		Fig. 2. Administration of HEL105–120 in saline abrogates the generation of cytokine-producing HEL105–120-specific cells upon challenge with HEL105–120 in CFA. HEL105–120 in saline (HEL105–120 treated) or saline (non-treated) was administered i.v. to BALB/c mice on day 13 (300 µg peptide), day 8 (100 µg peptide) and day 3 (100 µg peptide) before challenge. On day 0 mice were challenged with a standard dose of 7 nmoles (13.35 µg) of HEL105–120 in saline emulsified 1:1 with CFA. Enumeration of peptide-specific IFN-{gamma}-, IL-2- and IL-4-secreting cells present in the spleen of individual mice (A and B) or in pooled draining lymph nodes (C and D) was performed on day 6 or 10 after immunization. Panels (A and C) represent observations from two experiments and (B and D) represent observations from three experiments, each employing three mice per group per experiment. Each symbol corresponds to the mean number of antigen-dependent spots generated in triplicate ELISPOT wells used to enumerate CSC. Lines represent the mean of means ± SEs. Mice in control groups were immunized with saline/CFA and did not generate HEL105–120-specific cytokine-secreting spleen or LN cells (n = 5). Plotted data represent antigen-dependent responses, which were significantly greater than responses in antigen– wells (P < 0.05).

 
We then examined the consequences of eliminating HEL105–120-specific CD4 T cells, via the i.v. administration of HEL105–120 in saline, on the CD4 T cell response to other HEL peptides, normally generated upon immunization with ha-HEL. We used ALUM, a non-bacterial adjuvant free of detectable endotoxin, to increase the immune response to ha-HEL, as this allows us to more readily detect cytokine-secreting CD4 T cells specific for minor peptides (30). The results from three independent experiments (EX.1–3), employing 3–4 mice per group, are shown in Fig. 3. It is clear that pre-exposure to the major peptide virtually ablates the response to the major peptide and also dramatically reduces the CD4 T cell response to three minor peptides included in this analysis and normally generated upon HEL challenge. In particular, the generation of IL-2-, IFN-{gamma}- and IL-4-producing T cells, specific for the major minor peptide, HEL11–25, was greatly decreased. Statistical analysis of the pooled results from all experiments performed is shown in Fig. 4. This analysis shows that the activation of CD4 T cells specific for ‘minor’ peptides to be dependent on the presence of CD4 T cells specific for HEL105–120. The analysis of the CD4 T cell response to a large number of HEL peptides (see Fig. 4 and ref. 30) shows that the lack of response in HEL105–120 pre-treated mice, and evident from the observations of Figs 3 and 4, is not due to a shift in the specificity of the response to other peptides. We also extended our observations to examine responses at day 14 after HEL immunization to ensure that the lack of response to minor peptides, seen on day 10, is not due to a shift in the kinetics of the response. Responses to minor peptides at day 14 following HEL immunization were also barely detectable, mirroring the day 10 results (data not shown). We infer from these observations that the generation of minor peptide-specific CD4 T cells producing IL-2, IFN-{gamma} and IL-4 are CD4 T cell cooperation dependent. Alternatively, administration of HEL105–120 may generate HEL105–120-specific regulatory CD4 T cells that prevent the generation of minor HEL peptide-specific cells normally arising following HEL challenge.


Figure 3
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  		Fig. 3. Activation of CD4 T cells specific for one HEL peptide, following HEL challenge, operationally depends on functional CD4 T cells specific for other HEL peptides. HEL105–120 in saline (HEL105–120-treated) or saline (non-treated) was administered to BALB/c mice on days 13, 8 and 3 before challenge, as described in the legend to Fig. 2. On day 0, mice were challenged with 100 µg ha-HEL/ALUM. On day 10, spleen cells from individual mice were assessed for the presence of IFN-{gamma}-, IL-2- and IL-4-producing cells in response to the indicated HEL peptides using the ELISPOT assay. Observations from three experiments are shown. The data is plotted as a ‘stacked column’, in which responses to individual peptides from the same mouse are stacked on top of one another. Plotted data represent antigen-dependent responses in antigen+ wells, which were significantly greater than responses in antigen– wells (P < 0.05). Mice immunized with saline/ALUM did not generate HEL- or peptide-specific responses.

 

Figure 4
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  		Fig. 4. Statistical analysis of HEL peptide responses in HEL105–120 peptide-treated versus non-peptide-treated mice following challenge with ha-HEL/ALUM. t-tests were performed on pooled responses to the indicated HEL peptides from all HEL105–120 peptide-treated (‘T’, squares) and non-peptide-treated (‘N’, circles) mice, which subsequently received a challenge with ha-HEL/ALUM. Each symbol corresponds to the mean number of peptide-specific spots generated in triplicate ELISPOT wells used to enumerate CSC from a single mouse. Plotted data represent antigen-dependent responses in peptide+ wells, which were significantly greater than responses in peptide– wells (P < 0.05). Line represents the mean of the means of the responses from individual mice. Each peptide was employed in assessing the response in at least four separate experiments employing three to four mice per group. (A) Analysis of IL-2-producing cells. For HEL1–18: n = 13 (N), n = 12 (T); for HEL18–33: n = 13 (N), n = 12 (T); for HEL11–25: n = 14 (N), n = 12 (T); for HEL30–53: n = 13 (N), n = 12 (T); for HEL46–61: n = 19 (N), n = 18 (T); for HEL74–96: n = 23 (N), n = 20 (T) and for HEL105–120: n = 23 (N), n = 20 (T). (B) Analysis of IFN-{gamma}-producing cells is shown for only HEL11–25 and HEL105–120 as cells specific for other HEL peptides could not be detected in any of the mice tested; n = 14 for N and n = 12 for T. (C) Analysis of IL-4-producing cells. For HEL1–18: n = 13 (N), n = 12 (T); for HEL18–33: n = 13 (N), n = 12 (T); for HEL11–25: n = 14 (N), n = 12 (T); for HEL30–53: n = 13 (N), n = 12 (T); for HEL46–61: n = 19 (N), n = 18 (T); for HEL74–96: n = 23 (N), n = 20 (T) and for HEL105–120: n = 23 (N), n = 20 (T). The responses to HEL105–120 are separated from minor peptide responses to allow for closer analysis of numbers of minor peptide-specific spots. Experiments employing ha-HEL that had not been depleted of contaminating LPS produced similar results. *P = 0.01–0.05; ***P = 0.002–0.009, ****P < 0.0001, for responses to the indicated peptide in T versus N mice.

 
Administration of HEL105–120 i.v. in saline does not lead to the generation of functional HEL105–120-specific regulatory T cells
Administration of high doses of purified peptides i.v. in saline has been shown to result in the deletion of peptide-specific T cells rather than the generation of regulatory T cells (39–41). However, administration of peptides by other means and via other routes has been shown to generate T cells with regulatory properties (41–43). We therefore thought it prudent to explore whether regulatory T cells functionally exist in this system. Saline-treated mice or mice treated with HEL105–120 i.v. in saline were subsequently challenged with HEL coupled to OVA. The T cell response to OVA, HEL105–120, and to minor HEL peptides, i.e. the T cell response to HEL protein minus the response to the major peptide, was assessed 10 days following HEL–OVA challenge in six independent experiments, and the collated observations are shown in Fig. 5. These results clearly show that the number of IFN-{gamma}-, IL-2- and IL-4-secreting CD4 T cells, generated against OVA and the minor HEL peptides, are the same in saline and HEL105–120-treated mice. These results strongly suggest that HEL105–120 treatment does not generate HEL105120-specific regulatory cells capable of preventing the generation of OVA- or HEL minor peptide-specific CD4 T cells and that the generation of HEL minor peptide-specific CD4 T cells producing IFN-{gamma}, IL-2 and IL-4 are most probably CD4 T cell cooperation dependent. In contrast, cytokine responses to HEL105–120 were significantly diminished in all mice tested, confirming the inference that exposure to the single peptide without adjuvant leads to the inactivation of HEL105–120-specific T cells that is stable in the face of an immunogenic challenge. We also infer that new CD4 T cells specific for the major peptide are not generated in and emigrate from the thymus during the course of the experiment in sufficient numbers to result in a substantial CD4 T cell response on HEL–OVA challenge.


Figure 5
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  		Fig. 5. Administration of HEL105–120 i.v. in saline does not lead to the generation of functional HEL105–120-specific regulatory T cells. In six experiments, two to three BALB/c mice were treated with HEL105–120 in saline i.v. (‘T’, squares) or saline alone (‘N’, circles) on days 13, 8 and 3, as described in the legend to Fig. 2. On day 0, mice were injected with 100 µg of ha-HEL-OVA/ALUM i.p. Ten days later, spleen cells were plated in the optimized ELISPOT assay to separately enumerate specific cells producing IL-2, IFN-{gamma} and IL-4. Responses to HEL105–120, HEL minor peptides (response to HEL minus response to HEL105–120), and to OVA were enumerated. Plotted data represent antigen-dependent responses, which were significantly greater than responses in antigen– wells (P < 0.05). Each data point represents the spleen cell response in an individual mouse. The total number of mice in each group was as follows. HEL105–120 responses: for enumeration of IL-2-, IFN-{gamma}- and IL-4-secreting cells, n = 17 (N), n = 18 (T); for enumeration of HEL-specific CSC other than HEL105–120-specifc cells: for IL-2-secreting cells, n = 17 (N), n = 18 (T) and for IFN-{gamma}- and IL-4-secreting cells, n = 11(N), n = 12 (T); for enumeration OVA-specific cytokine-producing cells: for IFN-{gamma} and IL-2, n = 14 (N), n = 15 (T) and for IL-4, n = 17 (N), n = 18 (T). ***P < 0.0005 between HEL105–120 pre-treated (T) or non-peptide-treated (N) animals in response to the indicated antigen.

 
Generation of immune responses to sub-optimal doses of HEL requires physical linkage with OVA
The detection of minor peptide-specific T cells in HEL105–120-treated mice following HEL–OVA challenge strongly suggests that the physical coupling of HEL to OVA allows OVA-specific T cells to help the generation of minor peptide-specific T cells that are otherwise not generated following challenge with HEL alone, compare the responses in peptide-treated mice in Figs 4 and 5. Thus, administration of HEL–OVA recruits sufficient numbers of T cells to allow T cell cooperation and the generation of minor HEL peptide-specific cells. We wished to test the requirement for linkage in the generation of an immune response in which T cell cooperation appeared otherwise insufficient to generate CSC. Under conditions where the amount of HEL administered is very low, 1 µg, and resulting in non- or barely detectable responses, the co-administration of OVA only optimally helps the anti-HEL response if OVA is linked to HEL, compare responses to ha-HEL + ha-OVA versus responses to ha-HEL-OVA, shown in Fig. 6. Thus, the ‘help’ provided by OVA in the generation of the HEL response involves a mechanism that requires linked recognition. The marginal increases seen when ha-HEL and ha-OVA are administered separately is likely due to the in situ formation of a small amount of conjugate in the peritoneal cavity. Mixing sol-HEL and sol-OVA together results in visible precipitation, and we infer these proteins have a tendency to naturally co-aggregate and precipitate (D. R. Kroeger, unpublished observation).


Figure 6
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  		Fig. 6. Generation of immune responses to sub-optimal doses of HEL requires physical linkage with OVA. In two separate experiments, three to four BALB/c mice per group were given i.p. One microgram of heat-aggregated HEL (ha-HEL), 1 µg ha-HEL with 3 µg heat-aggregated OVA together (ha-HEL + ha-OVA) or 4 µg heat-aggregated HEL–OVA (ha-HEL–OVA) precipitated onto ALUM. Six days later, spleen cells were plated in the optimized ELISPOT assay to enummerate HEL-specific CSC. Each data point represents the spleen cell response of a single mouse; *P < 0.05, **P < 0.005 and ***P < 0.0005.

 
Examination of whether anti-CD40 mAb obviates the need for CD4 T cell cooperation in the activation of CD4 T cells
Our observations suggest that CD4 T cells specific for the minor epitopes of the HEL protein require help from CD4 T cells specific for HEL105–120 to be activated. In addition, CD4 T cells specific for peptides derived from the same cognate antigen appear to help CD8 T cell activation by ‘licensing’ APC (44, 45), and this function can be replaced by administration of an agonist anti-CD40 antibody (44, 45). We therefore investigated whether the underlying mechanism of CD4 T cell cooperation is also mediated by APC licensing via CD40–CD40L interactions. We injected normal mice and mice in which the HEL105–120-specific CD4 T cells had been ablated with 100 µg i.v. of either FGK4.5 or the isotype-matched control antibody, 2A3. We assessed, 10 days after immunization with ha-HEL/ALUM, the number of HEL105–120- and HEL11–25-specific CSC in the spleen as shown in Fig. 7. We found that the tolerance to HEL105–120 is stable upon anti-CD40 administration, i.e. no HEL105–120 specific CSC are generated in tolerant mice treated with FGK4.5 or 2A3. Importantly, the co-administration of anti-CD40 agonist does not result in the significant generation of HEL11–25-specific cytokine-secreting CD4 T cells. We conclude that stimulation through CD40 alone does not obviate the need for cooperating CD4 T cells in the generation of minor HEL peptide-specific responses.


Figure 7
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  		Fig. 7. Signaling through CD40 does not replace the requirement for T cell cooperation. Three to five BALB/c mice per group were treated with either saline alone (non-peptide-treated ‘N’; open circles) or HEL105–120 in saline (peptide-treated ‘T’; open squares) i.v. as described in Fig. 2. On day 0, mice were injected with 100 µg of ha-HEL/ALUM i.p. At the same time, mice were given 100 µg of either an isotype-matched control antibody (2A3) or an anti-CD40 agonist antibody (FGK4.5) i.v. Ten days later, spleen cells were plated in the optimized ELISPOT assay to separately enumerate specific cells producing IL-2, IFN-{gamma} and IL-4. Antigen-dependent cytokine spots per spleen are plotted. The pooled results of two independent experiments are shown; ns, not significantly different, *P < 0.05, **P < 0.005 and ***P < 0.0005.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Our observations demonstrate that the administration of the major HEL peptide, HEL105–120, abrogates the CD4 T cell response to this peptide, and other HEL peptides, on a subsequent HEL challenge. There are two conceivable ways of accounting for this finding. The first is that the efficient activation of CD4 T cells specific for non-major peptides requires CD4 T cells specific for the major peptide, a conclusion that supports the idea that CD4 T cell cooperation facilitates or is required to activate CD4 T cells to produce CSC. Alternatively, the administration of HEL105–120 could result in the generation of peptide-specific regulatory T cells that prevent the generation of cytokine-producing CD4 T cells specific for other peptides derived from the same antigen. This possibility should be considered in light of the existence of antigen-driven regulatory T cells (41–43). The regimen of peptide administration we used in our attempt to inactivate CD4 T cells specific for the major peptide is known to result in the physical disappearance of most of the progeny of the peptide-specific, peptide-stimulated CD4 T cells (39–41). The few CD4 T cells remaining appear to be anergic (see Fig. 2) and do not have detectable suppressive activity, even when responses to a second peptide are assessed following presentation of both peptides by the same APC (40, 41). In addition, we show that, following challenge with a heat-aggregated HEL-OVA conjugate, the total number of IFN-{gamma}-, IL-2- and IL-4-producing CD4 T cells specific for minor HEL peptides, and for OVA, are the same in mice pre-treated with either HEL105–120 or saline (see Fig. 4). This result demonstrates that prior administration of HEL105–120 did not generate T regulatory cells capable of preventing the generation of OVA or minor HEL peptide-specific T cells. The only way our observations could be accounted for in terms of the generation of T regulatory cells is if the suppressive activity of such cells was overcome following HEL-OVA challenge but not following HEL challenge. However, it is unlikely that the suppressive activity is all or none and, if Treg cells exist, they should exert some influence on the outcome of HEL-OVA challenge. Our results showing equivalent responses to OVA and minor HEL peptides following HEL-OVA challenge, as well as the findings of others (40, 41), do not support this possibility.

We conclude that CD4 T cell cooperation is required for CD4 T cell activation. The ability of OVA to optimally help anti-HEL CD4 T cell responses required, in a closely related experimental system, that the OVA antigen be physically linked to the HEL antigen, see Fig. 6. We envisage that this requirement for linkage for optimal CD4 T cell cooperation reflects the need for an APC to present both OVA and HEL peptides in mediating the cooperation between OVA- and HEL-specific CD4 T cells.

How can APC mediate CD4 T cell cooperation? An intriguing possibility is that, just as activated CD4 T cells can help in the activation of naive CD8 T cells by licensing APC, involving an interaction between CD40 on the APC and CD40L on the activated CD4 cell (44, 45), activated CD4 T cells may also be able to license APC to activate a naive CD4 T cell (23). However, unlike the licensing of APC to activate CD8 T cells (44, 45), we found that the requirement for CD4 T cell cooperation could not be replaced by CD40 signaling on APCs, see Fig. 7. We consider it likely that CD40–CD40L interactions may be required for CD4 T cell cooperation but that this interaction alone does not license APC to activate naive CD4 T cells. Additional signaling pathways, such as those provided by cytokines, are potential mechanisms by which CD4 T cell cooperation may be mediated and experiments are currently underway to assess the role of these pathways.

Direct tests of a critical role of microbial/danger signals in the activation of naive CD4+ T cells are difficult without a complete list of the microbial products or dangerous circumstances able to potentially generate this signal. However, we have shown that the very same antigen preparation, ha-HEL/ALUM, consistently generates cytokine-producing CD4 T cells specific for minor HEL peptides, including HEL11–25, in mice pre-exposed to saline, but not in mice pre-exposed to the major HEL peptide, HEL105–120. The CD4 T cell response to minor HEL peptides, observed in normal mice upon immunization with HEL, must mean, in the context of Microbial/Danger Signal Models, that our antigen preparation, or its mode of delivery, is able to generate the microbial/danger signal. The lack of significant activation of CD4 T cells specific for these same peptides, in mice pre-treated with the major peptide, on challenge with the very same antigen preparation, is paradoxical within the context of Microbial/Danger Signal Models. In this regard, a recent report shows that a major adjuvant function of alum occurs through the formation of uric acid (46). Our observations reveal that this formation of uric acid does not bypass the requirement for CD4 T cell cooperation in the activation of HEL-specific CD4 T cells.

We conclude that microbial/danger signals, of the kind envisaged in Microbial/Danger Signal Models, are most probably not required to activate CD4 T cells. We acknowledge the evidence that innate mechanisms of defense modulate adaptive immune responses and the potential importance of such modulation in the context of infectious disease (18, 20). However, we think it plausible that such modulation normally acts in the context of a separate, underlying immunological mechanism of CD4 T cell cooperation controlling whether responses are initiated, as proposed in the Two-Step, Two-Signal Model of CD4 T cell activation (23). We have argued elsewhere that a highly specific immunological mechanism controlling initiation of immune responses can provide more stringent control than that afforded by innate mechanisms (23, 24).

The striking nature of the observations reported here, in which the entire or almost the entire HEL-specific response is ablated following the inactivation of CD4 T cells specific for a single HEL peptide, might well be considered surprising, even within the context of a model for CD4 T cell activation that depends upon CD4 T cell cooperation. These observations are expected only if the number of HEL-specific CD4 T cells is barely sufficient, in a normal mouse, to obtain the required CD4 T cell cooperation to initiate an immune response. Our observations that the generation of responses to HEL administered without adjuvant required aggregation of the protein, and a highly sensitive ELISPOT assay to detect this response, support this possibility that the number of CD4 T cells is barely sufficient. These considerations provided us with the impetus to test the requirement for CD4 T cell cooperation in this experimental model.

The nature of the cooperating CD4 T cell is unclear. One possibility is that activated CD4 T cells are required to allow antigen to activate naive CD4 T cells, an assertion supported by older (22, 23) and more recent studies (47). These observations, if indeed the conclusion drawn from them is correct, raise the question of how the first CD4 T cells are activated, a question referred to as the priming problem (reviewed in refs 21, 22). Alternatively, T cell cooperation may occur between naive cells that reach a critical mass around a single APC, resulting in activation. The recent availability of a system, to detect the activated CD4 T cells required to initiate immune responses (47), should allow analysis of the various possibilities for how the first CD4 T cells are activated.

We draw three conclusions from these studies. (i) Substantial immune responses to non-microbial, protein antigens can be achieved when antigen is given without microbial adjuvant, and such responses are likely to be independent of contaminating LPS. (ii) The generation of IL-2-, IFN-{gamma}- and IL-4-producing CD4 T cells depends upon CD4 T cell cooperation when mice are challenged with antigen administered without microbial adjuvant and that this CD4 T cell cooperation is not mediated by CD40-CD40L interactions alone. (iii) Our observations are difficult to reconcile with strict forms of Microbial/Danger Signal Models and reinforce the paradox, within the context of such models, that sterile alloantigens can be immunogenic (23).

The implications of these findings are likely to be diverse, due to the central role CD4 T cells play in initiating and regulating immune responses. They are clearly relevant to understanding mechanisms by which organ-specific autoimmunity might be precipitated by exposure to foreign antigens that cross-react with self, which would include virally infected self cells. Cooperation between CD4 T cells in the activation of CD4 T cells explains the phenomenon of epitope spreading, seen in autoimmune disease, in which the existence of CD4 T cells specific for one epitope of a protein or cellular antigen leads to the activation of CD4 T cells specific for other epitopes (48). Our findings are also pertinent to vaccine design against tumors and infectious agents, as well as the design of strategies to achieve transplantation tolerance, primarily because the role of CD4 T cell cooperation has not been considered in these settings. Finally, a knowledge of the requirements for the activation of CD4 T cells bear on other central questions, such as the nature of the ‘decision criterion’ that controls the Th1/Th2 phenotype of an immune response.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Canadian Institutes for Health Research (MOP-1412 to P.A.B.); Natural Sciences and Engineering Research Council of Canada (to P.A.B.).


   Acknowledgements
 
We are indebted to H. Bull, D. Hamilton, C. Havele, J. Lee, K. McKinstry, D. Sacks, T. Strutt and H. Tabel for comments on the manuscript. D.R.K. was the recipient of a Canadian Institute for Health Research Canada Graduate Scholarships Masters Award.


   Abbreviations
 
ALUM, aluminum hydroxide adjuvant
APC, antigen-presenting cells
CoS, co-stimulatory
CSC, cytokine-secreting cells
ELISPOT, enzyme-linked immune spot
HEL, hen egg lysozyme
ha-HEL, heat-aggregated hen egg lysozyme
i.p., intraperitoneally
LN, lymph node
sol, soluble
TLR, toll-like receptor

   Notes
 
Transmitting editor: A. Singer

Received 18 August 2008, accepted 6 August 2009.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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