Differential expression of the cytokine receptors for human interleukin (IL)-12 and IL-18 on lymphocytes of both CD45RA⁺ and CD45RO⁺ phenotype from tonsils, cord and adult peripheral blood

Abstract

The objective of this study was to demonstrate the variable expression of cytokine receptors on naive versus memory human CD4⁺ T cell subpopulations in tonsillar tissue, cord blood and adult blood. We prove that the receptors for both interleukin (IL)-12 and IL-18 are expressed exclusively on memory T cells. This observation was seen not only on the CD45RO⁺ memory T cells but also on a significant percentage of the CD45RA⁺, CD62L⁻, CD27⁻ and CCR7⁻ populations. Furthermore, CD45RA⁺ CD62L⁺, CD27⁺ or CCR7⁺ CD4⁺ T cells that expressed IL-12Rβ1 and IL-18Rα did not express CD31, a marker for recent thymic emigrants. We reveal that cord blood lymphocytes do not express IL-12Rβ1 whereas IL-18Rα expression was detected at low levels. Importantly, the IL-12Rβ2 signalling chain, which is absent in all resting T cells, was up-regulated in both CD45RA⁺ and CD45RO⁺ T cells as a result of stimulation with anti-CD3 and anti-CD28 in vitro. This observed up-regulation was, however, restricted to 80% of the total CD4⁺ population. Finally, a very small proportion of the CD4⁺ CD45RO⁺ tonsillar T cells expressed the IL-12 and IL-18 receptors, thereby establishing the differential expression of these receptors between peripheral and tonsillar memory T cell subpopulations.

INTRODUCTION

The development of lymphocyte subpopulations and their subsequent differentiation into the spectrum of functional effector cells is controlled critically by cytokines. In sites of inflammation, and in response to a pathogenic insult, dendritic cells and macrophages synthesize interleukin (IL)-12, IL-18 and interferon (IFN)-γ which results ultimately in the differentiation of antigen-stimulated naive CD4⁺ T cells into fully mature Th1 effector cells [1]. These cytokines exert their influence on lymphocytes through interactions with complementary receptors. Therefore, the spectrum of cytokine receptors expressed on the surface of a given T cell is not only an indication of the possible functional potential of this cell, but may also provide a useful tool for identifying T cell subpopulations [2]. In this work, we explore successfully the possibility of distinguishing memory T cells through the differential cell surface expression of their cytokine receptors.

We have shown previously that the IL-12 receptor and IL-18 receptor binding chains, IL-12Rβ1 and IL-18Rα, respectively, are expressed preferentially on CD4⁺ CD45RO⁺ and CD8⁺ CD45RO⁺ lymphocytes from adult peripheral blood. In addition, a small proportion of CD45RA⁺ T cells were also shown to express these receptor subunits [3]. This work has now been confirmed and extended to clarify the following areas.

Initially, it was necessary to establish whether the expression of the receptors for IL-12 and IL-18 are expressed on CD45RA⁺‘prememory’ and/or ‘reverted’ memory T cells [4]. We approached this by determining the level of expression of IL-12Rβ1 and IL-18Rα on CD4⁺CD45RA⁺ populations which were positive for CD62L or CD27 or CCR7, as these cell surface molecules have been shown to be good indicators of truly naive cells [4–7], and on CD4⁺CD31⁺ populations, as these lymphocytes contain higher levels of T cell receptor excision circles (TRECS) and are considered to be recent thymic emigrants [8]. We further studied the expression of these cytokine receptors on cord blood lymphocytes, seeing that these cells develop in a relatively naive environment.

Subsequently, we investigated the expression of the IL-12 and IL-18 receptors on the effector and/or central memory cell populations. This was approached by determining the expression of these receptors on tonsillar T cells, a population representative of peripheral lymphoid tissue, and on CD45RO⁺ CCR7⁺ and CD45RO⁺ CCR7^– CD4⁺ populations, as CCR7 is a lymphoid homing receptor that has been shown to characterize central memory and effector memory T cells, respectively [9]. Finally, we stimulated T cells with anti-CD3 and anti-CD28 in vitro and measured the relative expression of these two cytokine receptors on the total CD4⁺ T cell population and on the fraction destined to become the Th1 subpopulation.

In summary, we have established that the expression of the IL-12 and IL-18 receptors are expressed preferentially on CD45RA⁺ reverted and CD45RO⁺ effector memory CD4⁺ cells, and that upon T cell receptor (TCR) stimulation a significant proportion of both CD45RA⁺ and CD45RO⁺ cells, not destined to become Th1 cells, are refractory to stimulation via the TCR and do not up-regulate the IL-12 and IL-18 receptors.

MATERIALS AND METHODS

Samples

Heparinized blood was obtained from 22 healthy volunteers who gave informed consent. Tonsils (n = 15) were obtained at routine tonsillectomy (median age 10, range 4–34). The study was approved by the ethics committee of the Royal Free Hospital and Hospital Germans Trias i Pujol.

Isolation and stimulation of lymphocyte subpopulations

Mononuclear cells were separated by Ficoll Hypaque density gradient. Two aliquots of the cell suspension were incubated with either monoclonal anti-CD45RA or anti-CD45RO-coated MACS beads (Dako, High Wycombe, UK) according to the manufacturer's instructions (Milteny Biotec, Bisley, Surrey, UK). Total mononuclear cells and the depleted populations were suspended at 2 × 10⁶ cells/ml in supplemented medium and were stimulated with anti-CD28 (1 µg/ml) in 96-well plates coated with anti-CD3. Control cells were cultured without stimulation. Cells were removed from the cultures after 0, 24, 48 and 72 h, stained with appropriate antibodies and analysed by flow cytometry. Tonsillar cells were obtained by teasing the tonsillar tissue and separation of the subsequent cells by Ficoll Hypaque density gradient centrifugation, adjusted at 2 × 10⁶/ml and stained with the relevant antibodies.

Detection of cytokine receptors

Cytokine receptors were detected in whole blood by a direct immunofluorescence method using four fluorochrome combination of reagents and analysed using a FACSCalibur cytometer (BD Biosciences, Oxford, UK). Isolated cells were similarly stained with the relevant combination of antibodies.

The two subunits of the IL-12 receptor and the alpha chain of the IL-18 receptor were detected by staining with anti-IL-12 Rβ1, anti-IL-12Rβ2 (Pharmingen, Becton Dickinson, Oxford, UK) and anti-IL-18Rα monoclonal antibodies (R&D Systems, Abingdon, UK). T cells were distinguished by the surface expression of CD3. The CD3⁺ population was further stained with either anti-CD4 or anti-CD8 and with anti-CD45RA (FITC) (Becton Dickinson), anti-CD45RO (FITC) or anti-CD45RB (FITC) reagents (Dako) to assess their expression within the naive, primed and terminally differentiated populations. The naive CD4⁺ cells were measured by the co-expression of CD45RA (or lack of expression of CD45RO) and CD27, CD62L and CD31 (Dako). Central memory and effector memory CD4⁺ T cells were distinguished by the expression of CCR7 (Pharmigen) on CD45RO⁺ CD4⁺ T cells.

Immunohistochemistry

Tonsil biopsies were frozen in liquid nitrogen. Cryostat sections were fixed in ethanol followed by acetone and sections were stained with anti-IL-12 antibody (R&D Systems) or with anti-CD68 (Dako), a macrophage marker. This was followed by a secondary antibody labelled with horseradish peroxidase and developed with 3,3′diaminobenzidine tetrahydrochloride (DAB) (Dako), respectively, as directed by the manufacturers. Isotype- matched immunoglobulins were used as a negative control.

Statistical methods

Data were analysed with the PRISM statistical package and, if not stated otherwise, was distributed normally and expressed as mean ± s.e.m. (95% confidence interval). When comparing three or more groups, we used the anova Kruskal–Wallis test; when comparing two groups we used the T test or paired T test if the results were obtained from the same samples. Spearman's correlation test was used to test the relationship between two sets of data. All tests of significance were two tailed.

RESULTS

The IL-12Rβ1 and IL-18Rα were preferentially expressed on CD45RA⁺ and CD45RO⁺ memory populations

As shown in Table 1 and Fig. 1, the majority of CD4⁺ T cells expressing the IL-12Rβ1 and IL-18α chains were of the CD45RO phenotype. Nevertheless, 6·5% and 10% of CD4⁺ and CD45RA⁺ T cells in adult peripheral blood also expressed the IL-12Rβ1 and IL-18Rα subunits. These lymphocytes were distributed equally between the ‘naive’ CD45RA⁺ (CD62L⁺, CD27⁺ and CCR7⁺⁾ and the ‘memory’ CD45RA⁺ (CD62L^–, CD27^– and CCR7^–) populations (Fig. 2). The observed CD45RA⁺ populations defined by CD62L⁺, CD27⁺ and CCR7⁺ overlapped significantly, covering 87 ± 2·2%, 92 ± 2·3% and 87 ± 2·5% of the CD45RA⁺. As some CD45RA⁺ cells can also co-express CD45RO, we also analysed the expression of CD62L⁺, CD27⁺ and CCR7⁺ on the CD45RO^– populations with very similar results (Fig. 2). The IL-12 and IL-18 receptors were absent from the recent thymic emigrants, which were defined by their expression of CD45RA and CD31.

Table 1.

Relative expression of IL-12Rβ1 and IL-18Rα on CD4⁺ T cells expressing different CD45 isoforms from adult blood, cord blood and tonsil

	N	CD4	CD4 CD45RA	CD4 CD45RO
IL-12Rβ1
Adult	12	24·8 ± 2·28	6·5 ± 1·71	34·3 ± 3·80
		20·0–29·5	2·4–10·3	25·9–42·7
Cord	9	1·8 ± 0·32	1·81 ± 0·32	n.a.
		0·6–2·5	0·6–2·5
Tonsil	11	10·6 ± 1·16	8·8 ± 1·31	10·9 ± 1·65
		8·1–13·1	5·9–11·8	7·25–14·6
IL-18Rα
Adult	12	37·5 ± 2·12	10·8 ± 1·51	47·0 ± 2·32
		321–41·9	7·43–14·2	41·7–52·2
Cord	9	10·7 ± 2·39	10·7 ± 2·39	n.a.
		5·1–16·44	5·10–16·44
Tonsil	11	12·0 ± 26·2	13·0 ± 1·71	25·6 ± 3·95
		17·0–4·27	9·2–16·8	16·7–34·4

Data are expressed as mean ± s.e.m. and 95% confidence interval (italics): N, number of patients; n.a., non-applicable. Lymphocytes from adult circulating blood, cord blood and tonsils were stained to identify the expression of the cytokine receptors IL-12Rβ1 and IL-18Rα⁺ on ‘naive’ (CD45RA) and ‘memory’ (CD45RO) CD4⁺ T cells subpopulations as described in Material and methods.

Fig. 1.

Up-regulation of regulatory cytokine receptors IL-12Rβ1, IL12Rβ2 and IL-18Rα on CD3 gated T cell and isolated CD45RA⁺ and CD45RO⁺ subsets. (a) Comparative dot plots representing the expression of IL-12Rβ1 (CD212), IL-12Rβ2 and IL-18Rα on CD3⁺ peripheral blood T cells from adults. Cells were stimulated with anti-CD3 and anti-CD28 and subsequently harvested and analysed at 0 h (left panel), 24 h (centre panel) and 48 h (right panel), respectively. Cells were stained for CD3, CD4, IL-12Rβ1 or IL-18Rα or IL-12Rβ2 and analysed by flow cytometry as described in Material and methods. (b) Comparative graphs representing the expression of IL-12Rβ1, IL-12Rβ2 and IL-18Rα on CD45RA⁺ (dotted bars) and CD45RO⁺ (black bars) CD4⁺ T cells from adults and from cord blood. Cells were stimulated with anti-CD3 and anti-CD28 and harvested subsequently and analysed at 0, 24, 48 and 72 h (days 1, 2, 3 and 4, respectively). Cells were stained as above. The figure shows that there is an up-regulation of all three regulatory cytokine receptors in a proportion of the CD3⁺CD4⁺ CD45RA⁺ and CD45RO⁺ populations.

Fig. 2.

Distribution of IL-12Rβ1, IL-18Rα, CCR7 on CD45RA^+/CD45RO⁺ CD4⁺ lymphocytes. Top panels: comparative dot plots representing the expression of CCR7 on CD45RA⁺ (a) and CD45RO⁺ (b) CD4 lymphocytes. Bottom panels: comparative dot blots representing the distribution of IL-12Rβ1 (c) and IL-18Rα (d) on CCR7⁺ and CCR7^– populations on CD4⁺ CD45RA⁺ peripheral blood gated T cells from adults. Cells were stained for CD4, CD45RA or CD45RO and CCR7 (a,b) or IL-12Rβ1 or IL-18Rα, CCR7, CD4 and CD45RA (c,d) and analysed by flow cytometry as described in Material and methods. Note that a small proportion of CD45RA negative CD4⁺ cells express CCR7 (a,b) and that approximately 50% of these cells are also positive for the IL-12 and IL-18 receptors (c and d).

In the CD4⁺ CD45RO⁺ population, the number of IL-12Rβ1⁺ and IL-18Rα⁺ cells were 34% and 47%, respectively. The IL-12Rβ1⁺ cells accounted for 9 ± 1·5% of the central memory (CCR7⁺⁾ population and 19 ± 2·4% of the effector memory (CCR7^–) subset. The IL-18Rα subunit was, in contrast, expressed more widely and was detectable on 53 ± 3·9% and 57 ± 4·9% of the central and effector fractions.

Cord blood CD4⁺ CD45RA⁺ cells expressed reduced levels of the IL-12 receptor compared to that observed in the adult population while the levels of IL-18Rα remained similar

As expected, due to the relatively small numbers of CD45RO⁺ cells, the percentage of CD4⁺ T cells expressing either IL-12Rβ1 or IL-18Rα in cord blood was strikingly and significantly lower (P = 0·001) than the percentages observed in adult peripheral blood. Nevertheless, there were also differences in the levels of IL-12Rβ1⁺ cells, which were almost negligible (<2%) in the cord blood CD45RA⁺ population compared to the same population in adult blood (6 ± 1·7%). On the other hand, the percentage expression of IL-18Rα was similar to that seen in adult CD45RA⁺ lymphocytes (Table 1).

The majority of IL-12Rβ⁺ and IL-18Rα⁺ memory cells are found in the circulation rather than in the peripheral lymphoid tissues

Tonsillar cells also showed a statistically significant two- to threefold (P < 0·001) reduction in the proportion of IL-12Rβ1⁺ and IL-18Rα⁺ lymphocytes compared to adult blood populations. In contrast to cord blood, however, this decrease was due to the lower percentages of tonsillar cells expressing IL-12Rβ1 and IL-18Rα in the CD45RO⁺ populations not in the CD4⁺ CD45RA⁺ T cells. The percentage of lymphocytes positive for these receptors in tonsillar CD4⁺C45RA⁺ T cells was similar to that detected in the corresponding adult population (Table 1).

Tonsillar macrophages do not secrete detectable levels of IL-12

It is well documented that in response to an antigenic stimulation both macrophages and dendritic cells are able to synthesize IL-12 [1]. As a result, we investigated the relative expression of IL-12 in tonsillar tissue obtained from five tonsillectomies. In addition, we determined the presence of IL-12 in two tonsils obtained from two patients diagnosed with infectious mononucleosis at the time of tonsillectomy. We found that the IL-12 was not detectable in any of the tissues samples studied except for occasional granulocytes within the blood vessels, even though the CD68⁺ macrophages were present in normal numbers (Fig. 3). In the Epstein–Barr virus (EBV)-infected samples, where there was an increase in the number of macrophages, IL-12 was not detectable either. On the other hand, these macrophages contained apoptotic tingible bodies, thus illustrating their phagocytic capacity.

Fig. 3.

Lack of expression of IL-12 on tonsil macrophages. Frozen sections from a tonsil from a control (a,c) and a patient with acute mononucleosis (b,d) were stained by immunocytochemistry with anti-CD68 a macrophage marker (a,b) and IL-12 (c,d) as described in Material and methods. The figure shows the lack of IL-12 despite the presence of well-differentiated macrophages. On the other hand, a few neutrophils inside a blood vessel do express IL-12 (c) (magnification ×200).

The up-regulation of the IL-12 and IL-18 cytokine receptors after anti-CD3 and anti-CD28 stimulation was restricted to a subpopulation of CD4⁺cells

In order to test the potential of the IL-12R^– and IL-18R^– T cells to up-regulate these receptors after stimulation, mononuclear cells were stimulated in vitro with anti-CD3 and anti-CD28. Following this stimulation, there was partial up-regulation of IL-12Rβ1, IL-12Rβ2 and IL-18Rα in both CD4⁺ and CD8⁺ subpopulations (Fig. 1) as well as in the CD4⁺CD45RA⁺ and CD4⁺CD45RO⁺ subpopulations. In CD4⁺ T cells from adult peripheral blood, the number of IL-12Rβ1- and IL-18Rα-expressing cells increased up to 63 ± 6·1% and 70 ± 6·7%, respectively, at 72 h of culture. The IL-12Rβ2 chain was up-regulated after activation in a similar proportion of ‘naive’ (CD45RA⁺) cells to that detected for the IL-12β1 chain, but to a lesser extent in the ‘memory’ (CD45RO⁺) subset. Cord blood CD45RA⁺ cells also up-regulated these receptors with similar kinetics observed in adult peripheral blood (Fig. 1).

Interestingly in our in vitro experiments, approximately one-fifth of all T cells subsets were unable to up-regulate any of the receptors of interest when stimulated with anti-CD3 and anti-CD28.

DISCUSSION

The cytokines IL-12 and IL-18 play a pivotal role in the differentiation of naive T cells into Th1 effector cells by inducing the production of IFN-γ as well as other Th1-related cytokines [1]. In this work, we have determined the expression of the receptors for IL-12 and IL-18 on T cell subsets, focusing in particular on recent thymic emigrants, naïve, central memory and effector memory populations before and after TCR stimulation.

Our results might indicate that, in adult peripheral blood, the expression of IL-12Rβ1 and IL-18Rα is restricted predominantly to T cells with a memory phenotype including 50% of the small proportion of CD45RA⁺ cells that do not express CD62L, CD27 and CCR7, that accounts for approximately 5% of the total CD4⁺ CD45RA⁺ population [4–7]. It has been suggested that this population may contain memory cells with a CD45RA⁺ reverted phenotype [4]. Whether the remaining population of the CD4⁺ CD45RA⁺ lacking CD62L, CD27 and CCR7 are cells prone to becoming Th1 cells or belong to a Th2 lineage merits further investigation. Nevertheless, the fact that around 20% of the CD4⁺ T cells are unable to up-regulate IL-12Rβ1, IL-12β2 or IL-18Rα receptor subunits upon anti-CD3 and anti-CD28 stimulation in our in vitro experiments suggests that at least a proportion of the IL-12R^– and IL-18R^– cells may belong to a Th2 lineage [10], although these data needs to be confirmed with functional assays.

Consistent with the observation that Th1 regulatory cytokine receptors are mainly expressed on ‘memory’ T cells, and that these receptors are also absent from the CD45RA⁺ CD31⁺ recent thymic emigrants [8,11], the percentage of T cells that express the IL-12 receptor in cord blood was almost non-existent. However, upon TCR stimulation (anti-CD3 and anti-CD28), a proportion of cord blood CD45RA⁺ T cells up-regulated both chains of the IL-12R with kinetics similar to those observed in adult circulating cells. These data are relevant to the immunocompetence of cord blood T cells. These cells have been reported to be ‘naive’, but immunocompetent and secreting type-1 cytokines such as IFN-γ and TNF-α as adult lymphocytes [12] if stimulated appropriately [13]. Thus, the relative lack of CD45RO⁺ cells in cord blood [14] might account for the lower incidence of acute graft-versus-host disease in cord blood compared to that seen in bone marrow transplantation [15]. However, there may still be a risk of a graft-versus-host reaction even with cord blood transfer, as the CD45RA⁺ cells could be able to acquire a Th1 phenotype after antigenic stimulation and subsequently mediate a graft-versus-host reaction.

The lower proportion of IL-12R⁺ and IL-18R⁺ T cells observed in tonsil tissue compared to peripheral blood suggest clearly that these receptors are expressed in the effector memory population. This reinforces the idea that lymphoid tissues are not immunological effector sites. Indeed, we have found that in contrast with inflamed tissue, where local macrophages produce high levels of IL-12 and IL-18 among other inflammatory cytokines, tonsillar macrophages have very low levels of IL-12, even in acute viral infections such as EBV and despite their clear phagocytic role, as judged by the presence of tingible bodies and measured by in situ immunocytochemistry.

Furthermore, these data also suggest that there may be mechanisms that protect the lymphoid apparatus from inflammation, although these may be over-ridden during acute microbial infection of a draining area when transient painful lymphadenopathy occurs, a suggestion that is consistent with the view that effector immune responses are focused at sites of inflammation rather than in central lymphoid organs.

On the other hand, in inflamed tissues, the presence of receptors for Th1 cytokines might facilitate an immediate response to IL-12 and IL-18 stimulation. As the migratory capacity of Th1 cells is increased further by IL-12 [16], the local production of IL-12 by macrophages, dendritic cells and granulocytes [17] at the sites of inflammation might exacerbate the inflammatory reaction.

Although the cells expressing IL-12Rβ1 and IL-18Rα do not express the IL-12Rβ2 chain while in the ‘resting’ state within the circulation, it is likely that the recruitment of these cells into the lymphoid apparatus during an immune response will lead to increased numbers of T cells expressing a fully functional IL-12R after antigenic stimulation. Upon stimulation, and confirming the data reported by others [18], the IL-12Rβ2 chain was up-regulated after activation in a similar proportion of ‘naive’ (CD45RA⁺) cells to that observed for the IL-12β1chain.

Finally, a significant proportion of both CD45RA⁺ CD45RO⁺ T cells are refractory to stimulation via anti-CD3 and anti-CD28, in our in vitro experiments as judged by their subsequent failure to up regulate IL-12R and IL-18Rα. This may allow some of the other T cells to develop into Th2 or T regulatory cells, providing a mechanism to suppress inflammation and establish a balanced immune response.

In conclusion, differential cytokine receptor expression provides an advantageous tool to detect and characterize functional T cell subpopulations, in particular to discriminate between naive and memory Th1 cell subpopulations.