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Human CD38 and its ligand CD31 define a unique lamina propria T lymphocyte signaling pathway

SILVIA DEAGLIO^*, ROBERTO MALLONE^*, GERMANO BAJ^*, DANILO DONATI, GIORGIO GIRAUDO, FRANCO CORNO, SANTINA BRUZZONE, MASSIMO GEUNA, CLARA AUSIELLO^¶ and FABIO MALAVASI^*2

^* Laboratory of Immunogenetics, Department of Genetics, Biology and Biochemistry,
First Surgical Clinics and
Laboratory of Clinical Immunology, IRCC, are at the University of Torino Medical School, Torino, Italy;
Department of Experimental Medicine-Biochemistry Section, University of Genova, Genova, Italy; and
^¶ National Institutes of Health (ISS), Roma, Italy

²Correspondence: Department of Genetics, Biology and Biochemistry, via Santena 19, 10126 TORINO, Italy. E-mail: malavasi{at}molinette.unito.it

SPECIFIC AIMS

CD38is a pleiotropic surface molecule endowed with ecto-enzymatic activities and simultaneously with receptorial functions, delivering non-lineage-restricted messages. The intrinsic ineptitude of the molecule to transduce signals is believed to be bypassed by establishing lateral associations with molecular complexes specialized in signaling. This hypothesis was initially demonstrated in circulating T cells, where CD38 is functionally dependent on the presence of an efficient TCR/CD3 complex. To further validate the working hypothesis of a molecular parasitism, we examined lamina propria (LP) T cells, a unique population of residential T lymphocytes, where the TCR/CD3 complex is present but is functionally impaired. Experimentally, the CD38-mediated signals of LP T lymphocytes were compared with those obtained by isolating circulating T cells of the same donors.

PRINCIPAL FINDINGS

1. Expression, structure, and enzymatic features of LP CD38
CD38 was highly expressed by CD45RO⁺ T cells (90%), with significantly higher intensity in the CD8 subset vs. CD4. The expression of the coreceptor molecule CD28 was clearly different from CD38, being prevalently detectable on the CD4 subsets. CD31, the only surface ligand so far identified for CD38, was almost constantly coexpressed with CD38.

LP CD38 displays the structural characteristics of the canonical surface molecule, as highlighted in a Western blot system using four different anti-CD38 mAbs.

Enzymatic characteristics of CD38 were comparatively assessed in circulating vs. LP T lymphocytes.

Although ADP-ribosyl cyclase and cADPR hydrolase activities were present in the two populations, differences in the cyclase/hydrolase ratio were observed, the cyclase being quantitatively prevalent in LP T cells.

2. CD38 ligation fails to induce Ca²⁺ modulation in LP T cells, at variance with circulating T, B, and NK cells
CD38 ligation was reported to influence Ca²⁺ fluxes in circulating lymphocytes as well as in the T cell line Jurkat. These effects were observed both with agonistic mAbs and with the CD31 ligand. No Ca²⁺fluxes could be detected in LP T cells upon ligation of the CD38 receptor, either using agonistic mAbs (Fig. 1 ) or engaging the molecule by means of L-CD31⁺ transfectants (not shown). The absence of Ca²⁺ fluxes could not be due to the laborious purification procedures, CD2 or CD3 ligations inducing Ca²⁺ mobilization with profiles similar to those of circulating cells (Fig. 1) . Nor could it be attributed to genetic polymorphisms, since PB and LP T cells were obtained from the same donor. As expected, CD3 proved the dominant Ca²⁺-mobilizing receptor in PB T cells, whereas CD2-induced fluxes were more sustained in LP T lymphocytes.

View larger version (89K): [in this window] [in a new window]   Figure 1. Ca2+ fluxes upon exposure of LP or PB T lymphocytes to anti-CD38, CD2 and CD3 mAbs. PB and LP T cells were purified from the same donors and loaded with the fluorescent indicator Fluo-3AM. After a preincubation (10 min at room temperature) with the relevant mAb, cells were analyzed continuously at 37°C using a FACSort. GAMIg was added 10 s after starting the analysis. Data are presented as density plot of the shift in the Fluo-3AM fluorescence over a 540 s time period. Data are from 6 experiments.

3. Analysis of phosphorylation of selected substrates upon CD38 ligation in LP and PB T cells indicates that a lack in PLC- activation is likely responsible for the block of Ca²⁺ mobilization
The absence of CD38-induced Ca²⁺ fluxes in LP T cells led us to evaluate the missing signaling step(s). The cells were tested for phosphorylation of PLC-, known to be involved in CD38-mediated Ca²⁺ signaling. CD38 binding in LP T lymphocytes was not followed by any increased phosphorylation of the PLC- substrate compared with the basal conditions (Fig. 2A , top panel). On the contrary, CD2 ligation was followed by PLC- phosphorylation. Comparative CD38 ligation in PB T lymphocytes induced consistent phosphorylation of PLC- substrate, similar to that triggered via CD2 (Fig. 2B )

View larger version (45K): [in this window] [in a new window]   Figure 2. PLC- phosphorylation in LP (A1) and PB (A2) T cells upon CD38 ligation. A1) LP T cells were treated for 1 min with the indicated mAb and lysed. After immunoprecipitation with anti-PLC- serum, the lysates were probed first with anti-PTyr mAb (right side) and subsequently with anti-PLC- mAb (left side). B–D) Western blot of tyrosine phosphorylated substrates on LP T cells after exposure to anti-CD38 and to control anti-CD2 mAbs. B) Anti-P-tyr mAb was used to probe an enriched preparation obtained by prior anti-P-tyr immunoprecipitation (left side). indicates the band identified by anti-lck mAb (right side). Arrowhead indicates an as yet unidentified phosphorylated substrate of 180 kD. HC = heavy chain of the precipitating mAbs. Gel run under reducing conditions. C) Western blot using polyclonal anti-LAT Ab on anti-PTyr immunoprecipitates (left side) and whole lysates (right side). D) Western blot using anti-syk serum on anti-PTyr immunoprecipitates (left side) and whole lysates (right side). NS = not stimulated.

CD38 ligation was followed by a consistent and reproducible phosphorylation of lck, as documented by a mobility shift highlighted on anti-pTyr immunoprecipitates, probed with anti-pTyr mAb and then with anti-lck mAb (Fig. 2C ). The degree of phosphorylation peaked at 2 min, decreasing at 3.5 min and maintaining stable levels at 5 min. Lck was also phosphorylated upon control CD2 ligation, although at a lower amplitude and with different kinetics. Peak values were reached within 3.5 min, becoming undetectable after 5 min (Fig. 2C ). A band of 180 kDa is currently under scrutiny. LAT phosphorylation was specifically induced via CD38 with a kinetics peaking at 3.5 min incubation (Fig. 2D ). The substrate phosphorylated upstream of LAT was subsequently identified as syk (Fig. 2D ).

4. Downstream analysis of CD38-mediated signals in LP: the block of Ca²⁺ mobilization does not hamper the synthesis and release of cytokines
Messages for IL-2, IL-4, IL-5, IL-6, IL-10, IFN-, and GM-CSF were found in LP T cells cultured with anti-CD38 mAbs. Anti-CD2 and anti-CD28 mAbs induced a wider spectrum of messages. The CD3 pathway was partly anergic, as witnessed by the low amounts of IL-10 and IFN- mRNA detected.

CD38 ligation also increases the secretion of IL-10 and IFN-, whereas IL-6 is only partially elevated.

5. Agonistic anti-CD38 mAb and the interactions between CD38 and the CD31 ligand yield similar signals, indicating that the CD38/CD31 interplay is preserved in a closed system as is the gut wall
To assess whether the CD38/CD31 interactions are also relevant in the LP microenvironment, cytokine assays were reproduced using CD31 as a trigger for CD38. L-CD31⁺ transfectants were cocultivated with LP T cells for 24 h. The mRNAs of IL-2, IL-4, IL-5, IL-6, IL-10, and IFN- were significantly increased and soluble IL-6, IL-10, and IFN- were quantitatively augmented.

CONCLUSIONS

Experience gathered so far indicates that the TCR/CD3 complex is necessary for the induction of CD38-mediated signals in circulating T cells. These observations support the hypothesis that CD38 specialized in parasitizing professional lineage-restricted molecules, e.g., BCR (B lymphocytes), CD16 (NK cells), and MHC Class II (monocytes). We extended our observation to normal T lymphocytes comparatively analyzed as circulating and as residential cells, the latter paradigmatically represented by those settled in the LP of the gut.

LP T lymphocytes are characterized by a partially anergic TCR/CD3, whereas the CD2 pathway is prominent and further enhanced by CD28 costimulation. The unexpected result of this work is that CD38 is operative in an environment where the TCR/CD3 complex is apparently refractory to signals, likely establishing new companions for its activities.

The receptorial features of CD38 are different from any model so far studied; the lack of Ca²⁺ mobilization is a unique finding among T cells. The physiological bases of this observation were investigated by testing CD38 ability to phosphorylate PLC-, the main ruler of Ca²⁺ mobilization. The results indicate that PLC- is phosphorylated in circulating T cells, whereas their residential counterpart displays an apparently refractory PLC- protein.

In spite of the blocked Ca²⁺, LP T lymphocytes phosphorylate distinct substrates after CD38 ligation. Indeed, lck, a member of the src family kinase was consistently phosphorylated after CD38 ligation. Next, we tracked the phosphorylation of syk and LAT. Signals transduced by several surface receptors rely on cascades involving lck activation and subsequent recruitment of kinases of the syk family. Such activated kinases are then available to phosphorylate substrates as the transmembrane adaptor protein LAT and—directly or indirectly—PLC-. CD38-mediated signaling shares with the above pathways several steps, at least up to LAT phosphorylation. From there on, the signals implemented by CD38 ligation exclude PLC- activation and, consequently, Ca²⁺ mobilization. The inference that could be extrapolated from the present results is that the block is after LAT activation and is incomplete, as witnessed by cytokine synthesis and release.

These results imply that CD38 signaling varies in T lymphocytes in function of the environment, the use of alternative pathways being a feature acquired during differentiation. Indeed, the majority of LP T cells display a CD45RO⁺ memory phenotype, reversing the situation described in the blood. Peripheral T cell commitment could be achieved by restricting the activation potential of T cells to fewer, selected pathways. A crucial observation is that CD38⁺ LP T cells interact with L-CD31⁺ transfectants, indicating that this receptor/ligand system is active also inside the tissues. The contacts between CD38⁺ LP T cells and CD31⁺ surrounding stromal, endothelial, and dendritic cells reproduce the biological signals obtained using agonistic mAbs.

The association of CD38 with distinct signaling receptors, its size and adhesive properties, suggest that the molecule might be part of the immunological synapse. Further, the ability of CD2 to remodel the membrane provides sufficient hints that such traffic includes someway CD38. Lateral associations of CD38 with CD2 and other receptors might modulate the exposure of different CD38 domains, altering the enzymatic and signaling performances. A reasonable inference of the last issue is that the associated molecule on LP T cells is not the CD3 complex. Further, altered cyclase/hydrolase ratio might indicate that the enzymatic functions modulate according to the environment (e.g., closed vs. open systems) or according to the life cycle (e.g., proliferation vs. apoptosis) of the cells. The reverse ratio between the cyclase and hydrolase activities may, however, also be attributed to structural variations of the molecule undetectable in Western blot or referable to its multimeric molecular organization.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of the signaling machinery exploited by CD38 in LP T lymphocytes, as compared to PB T lymphocytes. The results concerning PLC- phosphorylation, Ca2+ signaling, cytokine synthesis and release, and the interaction with the CD31 ligand are described in the present paper. Preliminary evidence suggests that CD38 exploits the signaling machinery of CD2 in the LP microenvironment.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0522fje ; to cite this article, use FASEB J. (January 19, 2001) 10.1096/fj.00-0522fje

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