Human mucosal CD4⁺ T cells but not blood CD4⁺ T cells respond vigorously towards CD28 engagement

Abstract

Human lamina propria T lymphocytes (LPT) possess functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). While they are characterized by a low proliferative response to T cell receptor (TCR)/CD3 stimulation in vitro their responsiveness to activation through the ‘co-stimulatory’ CD2-receptor is enhanced when compared to PBT. In this study, we demonstrate that engagement of another co-stimulatory receptor on both LPT and PBT, namely CD28, by a single monoclonal antibody (mAb), respectively, strongly activates the former but not the latter through a PI3-kinase dependent signalling pathway leading to the production of inflammatory cytokines such as interleukin (IL)-2, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF). In addition to the high sensitivity of LPT to CD2 stimulation, this finding supports the notion that ‘non-specific/innate’ mechanisms to activate T lymphocytes play a predominant role vis-à-vis‘TCR driven/adaptive’ responses in the intestinal mucosa. Furthermore, it suggests that results from preclinical tests for therapeutic antibodies performed with human blood derived T cells are probably insufficient to predict reactivities of tissue-resident immune cells, which – given their quantitative predominance – may critically determine the in-vivo response to such compounds.

Introduction

Adaptive immune responses are strongly down-regulated in the human intestinal mucosal microenvironment, a strategy that prevents immunization against the multitude of constantly existing foreign luminal antigens of nutritional and microbial origin and their rejection. Importantly, mucosal lamina propria T lymphocytes (LPT) are unable to proliferate in vitro towards T cell receptor (TCR)-directed stimuli, unlike their autologous peripheral blood T lymphocyte counterparts (PBT) [1],[2]. By contrast, LPT respond substantially stronger to in-vitro stimulation via the CD2 receptor than PBT with regard to both proliferation and cytokine production [1],[3]–[5]. This indicates an ‘alternative’ activation mode for T cells [6] residing in intestinal mucosal areas.

In this study, we examined whether the high responsiveness of LPT to TCR-independent stimuli also extends to another co-stimulatory receptor, CD28. Employing a single CD28 monoclonal antibody (mAb), the proliferative and cytokine responses of highly purified human CD4⁺ LPT and CD4⁺ PBT, respectively, to ligation of this receptor was analysed. Furthermore, the impact of CD28 engagement on the PI3-kinase/protein kinase B (AKT)/glycogen synthase kinase 3β (GSK-3β) pathway activation was determined comparing both cell populations.

Materials and methods

Reagents

The CD28 mAb clone 248 [immunoglobulin (Ig)M][7],[8] was obtained from the laboratory of Dr V. von Fliethner, Ludwig Institute, Epalinges, Switzerland, and was produced originally by A. Moretta (Genova, Italy). The antibody was used as hybridoma culture supernatant (IgM concentration as determined by nephelometry: 20 µg/ml) [9],[10]. The CD28 mAb clone CD28·2 (IgG1) was purchased from BD Bioscience (Heidelberg, Germany). The CD45 mAb clone AICD45·1/B220 (IgM) was produced in our own laboratory. It was applied as hybridoma culture supernatant (IgM concentration: 1·3 µg/ml). The CD3 mAb muromonab-CD3 (OKT-3) was obtained from the American Type Culture Collection (Rockville, MD, USA). Goat anti-mouse IgG and IgM were purchased from Dianova. LY294002, calyculin A as well as phospho-GSK-3β (Ser9), phospho-AKT (Ser473) and AKT-specific antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). GSK-3β mAb was obtained from BD Bioscience.

Tissues/samples

All human studies were approved by the ethics committee of the University of Heidelberg and were performed in accordance with the principles laid down in the Declaration of Helsinki. Informed consent was obtained from the patients. Gut specimens were derived from individuals undergoing resection for localized colon cancer or benign colonic diseases. Microscopically normal colonic mucosa was dissected from the surgical specimen near the resection margin and processed immediately for isolation of lamina propria cells.

Preparation of CD4⁺ T lymphocytes

Lamina propria mononuclear cells were isolated according to a modified method of Bull and Bookman [11], as described previously [12]. CD4⁺ LPT were purified using anti-CD4⁺ magnetic beads (Invitrogen, Grand Island, NY, USA). Briefly, lamina propria lymphocytes were incubated with anti-CD4 magnetic beads and separated from unlabelled cells using the MPC-L magnet (Invitrogen). Subsequently, beads and antibody were released from the cells by a polyclonal anti-Fab antibody specific for the CD4 antibody on the Dynabeads (Invitrogen) [purity >98% as determined by fluorescence activated cell sorter (FACS) analysis]. Peripheral blood was taken during the operation. Peripheral blood CD4⁺ T cells were obtained by Ficoll-Hypaque (GE Healthcare, Piscataway, NJ, USA) density gradient centrifugation and magnetic bead separation, as described above.

Experiments were performed with CD4⁺ T cells, as attempts to isolate pure populations of total lamina propria T cells remained unsuccessful. For preparation of CD45RO⁺ CD4⁺ PBT peripheral blood mononuclear cells were subject to negative magnetic cell separation using CD45RA MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) prior to purification of CD4⁺ T lymphocytes.

Stimulation of T lymphocytes

CD4⁺ PBT and CD4⁺ LPT were cultured in the presence of the CD28 IgM mAb (clone 248) or the CD45 IgM mAb (clone AICD45·1/B211) for the indicated time-periods. Both antibodies were added to the cells in soluble form. For CD3 stimulation, wells were coated with goat anti-mouse Ig/IgM (7·2 µg/ml) prior to incubation with the anti-CD3 mAb OKT-3 (0·05 µg/ml).

Proliferation assays

Cells (5 × 10⁴/well) were cultured in 96-well flat-bottomed plates (Nunc, Thermo Scientific, Rochester, NY, USA) for proliferation assays. Cells were pulsed with 1 µCi of [³H]-thymidine (GE Healthcare) at day 3 for 18 h and then harvested on a cell harvester (Inotec, Wohlen, Switzerland). [³H]-Thymidine incorporation was measured in a liquid scintillation spectrometer (Beckman Coulter, Inc., Indianapolis, IN, USA).

Gene expression analysis

CD4⁺ PBT and CD4⁺ LPT (5 × 10⁵) were collected in 300 µl lysis buffer and mRNA was isolated with the MagnaPure-LC device using the mRNA-I standard protocol. MRNA was reverse-transcribed using avian myeloblastosis virus reverse transcription (AMV-RT) and oligo-(dT) as primer (First-Strand cDNA synthesis kit; Roche Diagnostics, Mannheim, Germany), according to the manufacturer's protocol. Primer sets optimized for the LightCycler (RAS, Mannheim, Germany) were developed and provided by SEARCH-LC GmbH (Heidelberg, Germany). Polymerase chain reaction (PCR) was performed using the LightCycler FastStart DNA Syber Green I kit (RAS) according to the protocol provided in the parameter-specific kits. To correct for differences in the content of mRNA, the calculated transcript numbers were normalized according to the expression of the housekeeping gene cyclophilin B (CPB). Values were thus given as transcripts per 1000 transcripts of CPB.

Measurement of cytokine secretion

Cells (5 × 10⁴/well) were cultured in round-bottomed microtitre plates (Corning, Lowell, MA, USA) in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 2% l-glutamine and antibiotics as well as reagents, in a total volume of 200 µl/well. After 24 h, the supernatants were harvested, cleared by centrifugation and frozen at −80°C until assayed. The cytokine content of the supernatants was determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). The test was performed according to the manufacturer's instructions.

Preparation of cell extracts and Western blot analysis

Whole cell extracts of CD4⁺ PBT and CD4⁺ LPT were prepared as described previously [13]. Cell extracts were resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a polyvinylidene difluoride (PVDF) membrane (Pall, Port Washington, NY, USA) and stained with the appropriate antibody. Densitometric analysis was performed using a scanner (GS-800; Bio-Rad, Hercules, MA, USA) and Quantity One Software (Bio-Rad). The phosphorylation index was determined as follows: phospho-Akt or phospho-GSK-3β levels were normalized based on total Akt and GSK-3β levels, respectively. Phospho-AKT and phospho-GSK-3β levels after 1 h of CD28 stimulation of CD4⁺ LPT (Fig. 3b) or of CD45RO⁺CD4⁺ PBT (Fig. 3c) were set to 1. Phosphorylation levels at all other conditions were calculated as a fraction/multiple of 1.

Results

To investigate responses of human mucosal T lymphocytes (LPT) and blood lymphocytes (PBT), respectively, towards CD28 engagement, in this study CD4⁺ T lymphocytes were prepared from healthy intestinal mucosal tissue or autologous peripheral blood and subsequently subjected to stimulation by a single CD28 monoclonal antibody (mAb 248) at different concentrations. As shown in Fig. 1a,b, only LPT responded to CD28 engagement with the transcription of cytokine genes here exemplified by IL-2, TNF-α, IFN-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF). This differential response was not due to non-specific/unique properties of the IgM antibody independent of its CD28 binding capacity, because treatment of both cell populations with another IgM antibody (anti-CD45) at a concentration of 1·3 µg/ml, which clearly induced cytokine gene expression in LPT in the case of CD28 IgM, failed to elicit cytokine transcription (Fig. 1a). It was also not due to varying kinetics of gene expression in the two cell populations as determined in time–course experiments (data not shown). Furthermore, it cannot be explained by mere cross-linking of the CD28 molecule on the T cell surface: stimulation of both PBT and LPT, with another CD28 antibody (IgG1, clone CD28·2) in soluble form or cross-linked by goat anti-mouse Ig, which was precoated in the wells, did not induce cytokine gene expression in both cell populations (data not shown). Importantly, the differential reactivity of PBT and LPT to CD28 IgM activation is largely unrelated to their unequal proportions of CD45RO⁺ cells (PBT: ∼40% CD45RO⁺[14],[15]; LPT: ∼90% CD45RO⁺[16],[17]); while a minor increase in CD28 induced cytokine transcription could be observed in CD45RO⁺ PBT in comparison to total PBT, this increase was significantly lower than that observed in LPT when compared to PBT (Fig. 1b). In addition, to exclude the possibility that the lack of responsiveness of PBT towards mAb 248 was due to low viability, we performed stimulations with the combination of αCD3 +αCD28 (Fig. 1b and data not shown).

Fig. 1.

Stimulation by a single CD28 monoclonal antibody (mAb) induces cytokine production in lamina propria T lymphocytes (LPT) but not peripheral blood T lymphocytes (PBT). (a) Isolated CD4⁺ PBT and LPT were cultured in the presence or absence of different concentrations of the CD28 mAb 248 or of the CD45 mAb AICD45·1/B211 (1·3 µg/ml). After 4 h, mRNA expression of interleukin (IL)-2, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF) was determined by quantitative reverse transcription–polymerase chain reaction (rtPCR). Data are expressed as mean ± standard error of the mean. Shown are three independent experiments performed with cells from three different patients. (b) Isolated CD4⁺ PBT and LPT were cultured in the presence or absence of the CD28 mAb 248 [immunoglobulin (Ig)M; 5 µg/ml]. In separate experiments, CD4⁺ PBT and CD45RO⁺ CD4⁺ PBT were cultured under the same conditions. After 4 h, mRNA expression of IL-2, TNF-α, IFN-γ and GM-CSF was determined by quantitative rtPCR. Shown are 11 independent experiments performed with PBT and LPT from 11 different patients and five different experiments, respectively, performed with CD4⁺ PBT and CD45RO⁺CD4⁺ PBT isolated from five different volunteers. Lower right panel: isolated CD4⁺ PBT and LPT were subject to co-stimulation by the CD28 mAb 248 (5 µg/ml) and the CD3 mAb muromonab-CD3 (OKT-3) (0·05 µg/ml). TNF-α gene expression was determined after 4 h by quantitative rtPCR. Shown are five independent experiments performed with cells from five different patients. (c) Isolated CD4⁺ PBT and LPT were cultured in the presence or absence of the CD28 mAb 248 (5 µg/ml). Culture supernatant was harvested after 24 h, and the content of IL-2 and TNF-α was determined by enzyme-linked immunosorbent assay (ELISA) (detection limit 7·8 pg/ml and 16 pg/ml, respectively). Shown are three independent experiments performed with cells from three different patients.

Besides measuring gene expression we also determined IL-2 and TNF-α protein secretion by ELISA, which correlates with the PCR data (Fig. 1c). The interindividual variations with regard to the quantity of the detectable responses represent a well-known feature in human studies.

As shown in Fig. 2a, stimulation by the CD28 IgM antibody not only induced cytokine production but also cell growth in LPT, while PBT remained unresponsive. By contrast, PBT – but not LPT – proliferated following CD3 engagement, as shown previously [1],[2]. This differential response of PBT and LPT was also observed when a higher concentration of the CD28 mAb (10 µg/ml) and the CD3 mAb (0·5 µg/ml), respectively, was applied (Fig. S1). Again, the CD45RO⁺ phenotype of LPT only partially explains the selective induction of cell growth in these cells. Although CD45RO⁺ PBT proliferated more in response to CD28 IgM stimulation than total PBT, their proliferative response remained lower than that of LPT (Fig. 2b).

Fig. 2.

Stimulation by a single CD28 monoclonal antibody (mAb) induces proliferation in lamina propria T lymphocytes (LPT) but not peripheral blood T lymphocytes (PBT). (a) CD4⁺ PBT and LPT were cultured in the presence or absence of the CD28 mAb 248 (5 µg/ml) or the CD3 mAb muromonab-CD3 (OKT-3) (0·05 µg/ml) for 72 h. Proliferation was determined by [³H]-thymidine incorporation. Data are represented as mean ± standard error of the mean (s.e.m.) of triplicate cultures. Shown are six independent experiments performed with cells from six individual patients; n.d.: not done. (b) CD4⁺ PBT and LPT were cultured in the presence of the CD28 mAb 248 [immunoglobulin (Ig)M; 5 µg/ml] for 72 h (n = 6). In a separate set of experiments (n = 3; cells isolated from three different individuals), CD4⁺ PBT and CD45RO⁺CD4⁺ PBT were cultured under the same conditions. Proliferation was determined by [³H]-thymidine incorporation. Data are represented as mean ± s.e.m.

Given that we identified previously a role of PI3-kinase in mucosal inflammatory responses [12], we asked the question whether PI3-kinase would be also involved in the response of LPT to CD28 stimulation. Again, autologous mucosal CD4⁺ T cells and blood T cells were isolated and exposed to mAb 248. Lysates of stimulated and unstimulated cells, respectively, were prepared at different time-points after stimulation, subjected to gel electrophoresis followed by Western blotting with monoclonal antibodies specific for downstream targets of PI3-kinase (AKT; GSK-3β) in their phosphorylated forms, known to be indicative for PI3-kinase activity [18]–[24]. As shown in Fig. 3a, AKT phosphorylation on Ser 473 was already detectable 15 min after stimulation and increased further up to 1 h in lamina propria-derived T lymphocytes. In contrast, no or only a weak induction of AKT phosphorylation could be observed in PBT during this time-period, similarly to GSK-3β phosphorylation on Ser9. Figure 3b shows the phosphorylation index of AKT and GSK-3β as determined by densitometry. CD28-mediated phosphorylation of AKT and GSK-3β was not, or only marginally, increased in CD45RO⁺ PBT in comparison to the total PBT (Fig. 3c). This indicates that the observed differential phosphorylation of both kinases in PBT and LPT is unrelated to differences in the proportion of CD45RO⁺ T cells in both cell populations.

Fig. 3.

Enhanced CD28-induced phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3β (GSK-3β) in lamina propria T lymphocytes (LPT). (a) CD4⁺ peripheral blood T lymphocytes (PBT) and LPT were cultured in the presence or absence of the CD28 monoclonal antibody (mAb) 248 (5 µg/ml) for the indicated time-periods. The phosphorylation state of GSK-3β (Ser9) and AKT (Ser473) was determined by immunoblotting of whole cell extracts. Results are representative of three to four independent experiments performed with cells from three to four different patients. *Non-specific band. (b) Densitometric determination of the phosphorylation index of AKT (Ser473 and Thr 308; upper panels) and GSK-3β (Ser9; lower panel) in CD4⁺ PBT and LPT in response to CD28 activation. Data are expressed as mean ± standard deviation (s.d.) (n = 3–7; four independent experiments with cells from four different patients were performed for the medium control and the time-points 15 min, 30 min and 1 h; three separate experiments with cells from three different patients were performed for medium control and the time-points 1 h and 2 h; hence, there are n = 7 for medium control and the time-point 1 h, while for all other conditions there are n = 4 and n = 3, respectively). (c) CD4⁺ PBT and CD4⁺CD45RO⁺ PBT were cultured in the presence or absence of the CD28 mAb 248 (5 µg/ml) for the indicated time-periods. The phosphorylation state of GSK-3β (Ser9) and AKT (Ser473) was analysed by immunoblotting of whole cell extracts. Shown is the phosphorylation index of AKT (Ser473; upper panel) and GSK-3β (Ser9; lower panel) as determined by densitometry. Data are represented as the mean ± s.d. (AKT phosphorylation: n = 3 for all conditions using cells from three different individuals; GSK-3 phosphorylation: n = 4 for all conditions using cells from four different individuals).

In the next set of experiments we attempted to substantiate the central role of PI3-kinase in CD28 responses of LPT employing the PI3-kinase specific inhibitor LY294002 [25]. As shown in Fig. 4, cytokine gene expression and proliferation induced by CD28 antibody was inhibited strongly by LY294002. Although not shown, LY294002 also inhibited AKT and GSK-3β phosphorylation.

Fig. 4.

Inhibition of cytokine gene expression and proliferation by the PI3-kinase inhibitor Ly294002 in lamina propria T lymphocytes (LPT). CD4⁺ LPT were cultured in the presence or absence of the CD28 monoclonal antibody (mAb) 248 (5 µg/ml) for 4 h. Ly294002 was added 30 min prior to stimulation. (a) interleukin (IL)-2, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and granulocyte–macrophage colony-stimulating factor (GM-CSF) mRNA expression was determined by quantitative reverse transcription–polymerase chain reaction (rtPCR). Relative inhibition (in %) of cytokine gene expression by Ly294002 is shown. Data are expressed as mean ± standard deviation (s.d.) (n = 5; five independent experiments were performed with cells from five different patients); n.d.: not done. (b) Proliferation was determined by [³H]-thymidine incorporation. Relative inhibition (in %) of cell growth by Ly294002 is shown. Data are expressed as mean ± s.d. (n = 4; four independent experiments were performed with cells from five different patients).

Discussion

Human T lymphocytes are apparently able to employ various pathways for their activation, dependent on their respective environment. With regard to the intestinal mucosa, ‘innate’ triggering mechanisms that can be mimicked experimentally through exclusive ligation of CD28 (or CD2) by mAbs are likely to play a fundamental role.

Whether activation through the receptors CD28 or CD2 is involved in physiological as well as pathological responses in vivo remains a matter of speculation. Nevertheless, so far they represent the only known receptors through which T lymphocytes can be activated without concomitant triggering of the T cell antigen receptor complex [6],[26],[27]. With regard to triggering via CD2, combinations of two antibodies are invariably required for activation in vitro[6]; regarding CD28, so far only ‘superagonistic’ antibodies were known to possess this capacity in man as well as in several animal species when exposed to PBT [28].

The as yet known ligands for CD2 (CD58) and CD28 (CD80/CD86) are not expressed in the mucosal environment in its healthy state [29]–[31]. However, they are clearly induced on mucosal myeloid cells under inflammatory conditions [32],[33], where they may contribute to adaptive immunity associated with profound attack towards foreign luminal antigens. Whether they act in concert with as yet unidentified locally existing ligands to trigger CD28 (or CD2) mediated ‘innate’ immune responses remains to be investigated. At least with regard to CD2, it is well established that agonistic CD2 directed mAbs capable of activating human T cells do not block its CD58 binding site [6],[26].

Hypersensitivity of LPT to stimulation by mAb 248 is not caused by differential CD28 expression, as comparable levels of this molecule can be detected on the cell surface of both LPT and PBT (data not shown; [3]). It is also not due to non-specific effects of IgM unrelated to the CD28-stimulatory capacity of this antibody. Furthermore, it is largely unrelated to the CD45RO⁺ phenotype of LPT. With regard to the triggering mechanism by mAb, 248 mere cross-linking/multimerization of CD28 molecules on the T cell surface cannot serve as an explanation as (a) only LPT but not PBT (even at substantially higher antibody concentrations) are activated, and (b) another CD28 directed antibody (clone CD28·2) was inactive when cross-linked to plastic surfaces. This suggests that the activating capacity of the mAb 248 (in comparison to mAb CD28·2) may be due to the recognition of specific epitopes of the CD28 molecule by this antibody. Further characterization of signalling events under steady-state and activation conditions may provide insight into the molecular basis of the enhanced sensitivity of LPT to CD28 receptor ligation when compared to PBT.

The intestinal mucosa represents by far the largest accumulation of T lymphocytes in the human body. Moreover, the latter (LPT), when compared to circulating blood T cells (PBT), are more reactive to CD2 or CD28 triggering by some orders of magnitude, particularly with regard to cytokine secretion. Therefore, our present results may also provide additional hints as to the explanation of the severe, life-threatening cytokine storm that occurred in human volunteers to whom the superagonistic CD28 antibody, TG1412, was delivered [34].

Finally, this study suggests that standard preclinical tests of therapeutic antibodies performed with human peripheral blood-derived mononuclear cells may not necessarily predict the response of tissue-resident immune cells to such agents. Given their quantitative predominance, reactivity of tissue resident cells may significantly determine clinical responses to immunomodulatory drugs.

Disclosure

The authors declare that there is no conflict of interest.

Supporting information

Fig. S1. Stimulation by a single CD28monoclonal antibody (mAb) induces proliferation in lamina propria Tlymphocytes (LPT) but not peripheral blood T lymphocytes (PBT).CD4⁺ PBT and LPT were cultured in the presence orabsence of two different concentrations of the CD28 mAb 248[immunoglobulin (Ig)M; 10 and 5 μg/ml] or the CD3 mAbmuromonab-CD3 (OKT-3) (0·5 and 0·05 μg/ml) for 72h. Proliferation was determined by [³H]-thymidineincorporation. Shown are two independent experiments using cellsfrom two different patients. Data are represented as mean ± standard error of the mean of triplicate cultures.