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Technical Advance |
From the Departments of Pathology,*
Biomedical Sciences
and Biotechnology,
and
Pediatrics,
University of Brescia, Brescia,
Italy; the Queen Elizabeth Hospital,§
Hong
Kong, China; the University of Verona,¶
Verona, Italy, and the Section on Lymphocyte Signaling,||
Cell Biology and Metabolism Branch, National Institutes of Health,
Bethesda, Maryland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PLC-1) or creates sites of binding for
proteins involved in the activation cascade.1-3
Linker
for activation of T cells (LAT) is an integral membrane protein of
36–38 kd that plays an important role in linking engagement of the TCR
to the biochemical events of T cell activation.4,5
It is
one of the most prominent tyrosine-phosphorylated proteins after TCR
engagement.4,6
LAT is a substrate of activated ZAP-70 and
Syk PTKs and, on tyrosine phosphorylation, it binds Grb2,
PLC-1, the p85 subunit of PI3K, and other critical signaling
molecules, thereby recruiting these molecules to the plasma
membrane.7-10
Localization of these signaling molecules
to the membrane has several consequences. Phosphorylation of tyrosine
residues required for enzymatic activation is enhanced and formation of
protein complexes occurs.4
By analysis of RNA, LAT also
was shown to be expressed in NK and mast cells.4
Much less
is known about its function in these cell types. A rabbit polyclonal antibody, which was generated against the cytosolic portion of LAT,4 was used in this study to evaluate the immunohistochemical expression of LAT in normal and pathological hematolymphoid tissues. We also assessed the specificity of anti-LAT antibodies for the identification of T cells, tested its usefulness as an immunohistochemical reagent, and investigated its possible role in the study of lymphoid neoplasms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Paraffin-embedded normal lymphoid tissues included lymph nodes showing various forms of reactive changes (n = 10 cases), thymuses obtained during cardiac surgery or surrounding thymomas (n = 3), spleens removed after trauma or because of immune thrombocytopenia (n = 4), bone marrows (n = 6), and small intestine (n = 2). In addition, hematopoietic tissues from three embryos aged 11–12 weeks of gestation were analyzed. Freshly frozen samples of reactive lymph nodes (n = 3), spleen (n = 1), and thymus (n = 1) were also used.
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood after Ficoll-Hypaque gradient centrifugation and depleted of plastic adherent cells. For purification of polyclonal natural killer (NK) or T cell populations, PBMC were incubated with anti-CD3 monoclonal antibody (JT3A, gift of Dr. A. Moretta, University of Genova, Italy) for 30 minutes at 4°C, followed by treatment with goat anti-mouse-coated dynabeads (Dynal, Oslo, Norway) for 30 minutes at 4°C. The resulting CD3-negative lymphocyte populations, containing approximately 1% CD3+ cells, 20–30% HLADR+ cells, and 70–80% CD16+CD56+ cells, were cultured in rIL-2 (Cetus Corp., Emeryville, CA). To obtain polyclonally activated T cell-enriched lymphocyte populations, PBMC were stimulated with 0.1% (v/v) PHA (Gibco, Paisley, UK) for 24 hours and then cultured in rIL-2.
Neoplastic Tissues
Two-hundred and sixty-four cases of nodal and extranodal
hematolymphoid neoplasms were gathered from different institutions. All
neoplasms had been previously characterized immunophenotypically on
paraffin sections, and in many cases on frozen sections as
well (Tables 1–4)
.
All lymphomas were classified according to the International Lymphoma
Study Group Classification11
and included all major
subtypes of Hodgkin's and non-Hodgkin's lymphomas.
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Tissue samples had been fixed in various fixatives, including buffered formalin, B5, Bouin, and Hollande, and embedded in paraffin. Bone marrow biopsies were fixed in B5 for 3 hours and decalcified in 0.1 mol/L EDTA disodium salt aqueous solution for 2–8 hours. Fresh tissues were immediately frozen after biopsy in liquid nitrogen and stored at -80°C until used.
CD3+CD16-CD56- T cell and CD3-CD16+CD56+ NK cell populations were washed 3 times in 0.9% NaCl solution, resuspended at a concentration of 5 x 106 cells/ml, and used for cytospin preparations (100 µl/each slide). Slides were air-dried for 24 hours, then fixed in -20°C absolute ethanol for 30 minutes, dried, and used for immunocytochemical staining.
Immunostaining
Details on the production and characterization of the rabbit anti-LAT antibody are reported elsewhere.4 Immunostaining for LAT was performed on paraffin sections after antigen retrieval in microwave (3 boiling cycles, 5 minutes each, at 750 W, with an interval of 1 minute between cycles) in citrate buffer, pH 6.0. The polyclonal antibody anti-LAT was applied at a dilution of 1:800 in Tris-HCl buffer, pH 7.2–7.4, for 45 minutes and was followed by biotinylated anti-rabbit antibody (30 minutes) and peroxidase-conjugated streptavidin-biotin complex (30 minutes) (Bio-S.P.A., Milan, Italy). On cryostat sections, the procedure was similar but the microwave heating was avoided. Sections were air-dried for 18 hours and LAT was applied at a dilution of 1:100. On cytospins, the sample was subjected to microwave heating once.
In all cases of T-cell lymphomas and anaplastic large cell lymphomas (ALCL), serial paraffin sections were also stained with a polyclonal antibody anti-CD3 (Dako, Milan) (1:200; microwave antigen retrieval in 1 mmol/L EDTA buffer, pH 8.0, 2x 5' cycles). Furthermore, the cases of ALCL were also evaluated for their reactivity with the monoclonal antibody ALK1 (Dako) (1:10; microwave antigen retrieval in citrate buffer, 3x 5' cycles), which recognizes a formalin-resistant epitope in the nucleophosmin-anaplastic lymphoma kinase chimeric protein.12 Both CD3 and ALK1 immunostaining were performed using the same indirect immunoperoxidase technique adopted for LAT. Thymuses from embryos were also stained with polyclonal antibodies anti-CD3 and anti-TdT (Dako) (1:200; microwave antigen retrieval in 1 mmol/L EDTA buffer, pH 8.0, 3x 5' cycles, overnight incubation of the primary antibody).
Negative controls for both tissue and cytospin immunostainings were performed using either the preimmune serum or an irrelevant polyclonal antibody (anti-Hepatitis B virus core antigen, Dako) instead of anti-LAT.
Analysis of the distribution of LAT on PMBC was performed using two-color fluorescence cytofluorometric analysis (FACS) (Ortho Cytoron Absolute) as previously described.13 For FACS analysis of LAT in combination with monoclonal antibodies JT3A (IgG2a, anti-CD3), kd1 (IgG2a, anti-CD16), and C218 (IgG1, anti-CD56) (all antibodies provided by Dr. A. Moretta), membranes were permeabilized with 0.2% saponin (Sigma) in phosphate buffered saline (PBS), pH 7.6, for 5 minutes after fixation with 4% paraformaldehyde in PBS. As second reagents, fluorescein isothiocyanate-conjugated swine anti-rabbit antibody (Dako) and phycoerythrin-conjugated isotype-specific goat anti-mouse antibodies (Southern Biotechnology Associates, Birmingham, AL) were used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal Tissues and Cells
In the thymus, expression of LAT was identifiable throughout all
stages of thymocyte differentiation, including the large cortical
blasts (Figure 1, a and b)
. In embryos of
10–12 weeks' gestation, thymuses already showing a lobular
architecture contained lymphoid cells that expressed LAT (Figure 1c)
and CD3, but were negative for TdT (data not shown). In peripheral
lymphoid tissues, LAT-positive lymphocytes were located in the known
T-cell areas in lymph nodes and spleen (Figure 1d)
and the
immunoreactivity paralleled that obtained with anti-CD3 in both frozen
and paraffin sections. In the small intestine, intraepithelial T-cells
were also positive for LAT (Figure 1e)
. In bone marrow, LAT was
expressed by the sparse T lymphocytes present in interstitial spaces,
and also by platelets and megakaryocytes (Figure 1g)
that exhibited a
strong reactivity in the cytoplasm; all other hematopoietic cells were
negative. Reactivity for LAT was also noticed on tissue mast cells
(Figure 1h)
in the form of delicate plasma membrane and granular
cytoplasmic labeling.
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, epithelioid histiocytes, and
dendritic cells were completely negative for LAT. Similarly, LAT was
not expressed on endothelial cells and epithelia, including thymic
epithelium and Hassal bodies.
On PBMC, LAT was expressed, in addition to
CD3+ T cells, on resting NK cells
CD16+ and CD56+ (Figure 2a)
. As shown in Figure 2, b and c
, LAT
staining was also found on T and NK cells in culture with rIL-2; on
CD3-CD16+CD56+
NK cells purified from PBMC, LAT was obviously expressed, although the
intensity of reactivity was much weaker than that recognized on
purified CD3+ T cells. Cell labeling was
completely absent on cytospins stained with negative control antibodies
(Figure 2d)
.
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Hodgkin's and non-Hodgkin's Lymphomas
Neoplastic cells in all cases of Hodgkin's
(n = 15) and B-cell non-Hodgkin's lymphomas
(n = 100) (Table 1)
were negative for LAT, but
contained variable amounts of LAT-reactive nonneoplastic T cells
(Figure 3, a and b)
.
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. The intensity of
reactivity and the percentage of labeled cells varied among different
types of T-cell lymphomas. In lymphoblastic lymphomas all neoplastic
cells were strongly labeled by anti-LAT; in contrast, in peripheral
T-cell and NK/T-cell lymphomas the reactivity was intense in 28/53
cases (52.8%) and recognizable in the vast majority of cells in 50/53
(94.3%). LAT expression showed no substantial differences in
peripheral T-cell lymphomas with relationship to primary location, cell
morphology, and immunophenotype, with the exception of NK/T-cell
lymphomas, which were more frequently characterized by weaker and a
lower number of labeled cells.
The ALCL included 39 extracutaneous and 5 purely cutaneous cases.
On the basis of the expression of at least one T-cell marker (CD3, CD5,
CD8, CD43, CD45R0, TCR-ß) and no B-cell markers (CD20 or CD79a), 25
cases were classified as T-cell and 19 as null-cell phenotype. Fourteen
cases (31.8%) reacted with LAT; all were represented by extracutaneous
lymphomas. Ten of them were of T-cell phenotype and four were
null-cell. LAT-positive ALCL showed intense reactivity in 5/14 cases
(35.7%) and it was recognizable in the vast majority of cells in 10/14
(71.4%) (Figure 3h)
.
Comparison of LAT and CD3 Expression in T-Cell and NK/T-Cell Lymphomas Excluding ALCL
In T-lymphoblastic lymphoma, LAT positivity was more intense and
diffuse than CD3 (Figure 3, c and d)
. Among peripheral T-cell
lymphomas, only two LAT-negative cases were observed. One was a large
cell lymphoma presenting an aberrant phenotype with loss of CD3,CD5,
CD43, and TCR-ß. The other LAT-negative case was a NK/T cell lymphoma
that strongly expressed cytoplasmic CD3. Three cases of LAT-positive
cutaneous T-cell lymphomas did not express CD3 (two also lacked CD5),
but were positive for CD2, CD4, and TCR-ß.
Comparison of LAT, CD3, and ALK1 Expression in ALCL
The distribution of LAT, CD3, and ALK1 in ALCL is reported in
Table 3
. Seven ALCL cases were
LAT+CD3+, 7 were
LAT+CD3-, 9 were
LAT-CD3+, and 21 were
LAT-CD3-. Among ALCL
T-cell type (25 cases), CD3 labeled a higher number of cases (19/25;
76%) than LAT (10/25; 40%). Interestingly, LAT and CD3 expression in
ALCL T-cell type was discordant in a significantly higher number of
cases than in other peripheral T-cell lymphomas (12/25
versus 4/55, respectively; Fisher's exact test:
P < 0.001) (Figure 3, h and i)
. Immunostaining for
ALK1 was available in 42 cases, 10 of which showed nuclear ±
cytoplasmic positivity. Although the majority of ALK1-positive cases
were LAT-positive (6/10), in comparison with ALK1-negative cases (8/32)
this difference was not statistically significant (Fisher's exact
test: P = 0.059) (Figure 3j)
.
Hematopoietic Nonlymphoid Neoplasms
In chronic myeloproliferative disorders and myelodysplastic
syndromes, cytoplasmic LAT expression was consistently identified in
normal and atypical megakaryocytes, including the micromegakaryocytes
typically found in chronic myeloid leukemia and myelodysplasia (Figure 4, a and c)
.
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, the only
positive cases were two megakaryocytic leukemias, where the blast cells
were labeled in their cytoplasm. An additional case of chronic myeloid
leukemia in blastic transformation contained two cell populations, one
formed by myeloid blasts (myeloperoxidase and
CD34+) and one by megakaryocytic blasts,
expressing LAT, Factor VIII-related antigen, and CD34. In all pathological conditions, reactivity of LAT on megakaryocytes was strong and particularly helpful in the identification of abnormal forms; moreover, it was frequently stronger than that of other megakaryocytic markers on paraffin sections, such as Factor VIII-related antigen and CD61 (data not shown).
Four of five cases of mastocytosis showed LAT positivity, including 3/3
cases of cutaneous mastocytosis and 1/2 cases of systemic mastocytosis
(Figure 4d)
. As with normal mast cells, LAT expression in neoplastic
mast cells was finely granular in the cytoplasm and delicate on the
cell membrane, a pattern of staining that clearly differed from that on
T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-positive cells.16,17
According to our findings, CD16+ and
CD56+ NK cells from peripheral blood express LAT;
the same result was obtained on cytospins from
CD3-CD16+CD56+
NK cells and
CD3+CD16-CD56-
T cells in culture with r-IL2, where it was also obvious that NK cells
express less LAT than T cells. These observations confirm the data
obtained on YT-NK-like cells.4
Because NK cells
express TCR-
18
together with both Syk and
ZAP-70,19,20
and in view of what is known about these
molecules in T cells,21,22
it is very possible that LAT is
also involved along the TCR--Syk/ZAP-70 pathways in NK cells.
Expression of LAT in normal human mast cells was also shown. The fine
granular positivity in the cytoplasm and on the cell membrane on mast
cells is a distinctive cellular pattern of distribution, as compared to
T cells. Signaling pathways coupled to the Fc
receptor in mast cells
are quite similar to those involved in TCR-mediated signaling in T
cells.23
The Syk PTK is recruited to the receptor and a
number of molecules such as Grb2 and PLC-1 are involved in
intracellular pathways.4
It is likely that LAT has a
central function in these cells as well.
The strong reactivity of anti-LAT with human megakaryocytes and platelets is a novel finding. Mice genetically engineered to lack the adapter protein SLP-76, a protein known to bind LAT,24,25 develop a profound block in thymocyte development and demonstrate severe bleeding associated with thrombocytopenia.26 Moreover, it is well recognized that megakaryocytes express Syk and it has been shown that Syk-/- mice show severe hemorrhagic manifestations in utero and die shortly after birth, though they do not have a defect in platelet number.27,28 The detection of LAT in platelets and its known interactions with SLP-76 and Syk suggest that LAT may have a critical function in these cells.
The general pattern of reactivity observed in normal human tissues have been largely reproduced by studies in neoplastic counterparts. Among hematolymphoid neoplasms, anti-LAT stained the great majority of T-cell neoplasms (excluding ALCL), but staining was negative in all cases of B-cell lymphomas and Hodgkin's lymphomas. Anti-LAT did not label B-lymphoblastic lymphomas but strongly labeled T-lymphoblastic lymphomas. The reagent thus represents a valuable immunohistochemical marker for lineage assignment in lymphoblastic neoplasms. Existing antibodies reactive with antigens in paraffin samples have not been optimal for this purpose. For example, as also shown in this study the T-cell marker CD3 can be weak and focal,29,30 and UCHL1/CD45R0 is frequently negative;31,32 moreover, L26/CD20 is not uncommonly negative in B-cell lymphoblastic lymphomas33 and CD79a has recently been shown to stain a proportion of T-lymphoblastic tumors.34 Thus, LAT should help in determining the presence of a T-cell phenotype in lymphoblastic lymphomas. The observed expression of LAT on thymocytes at the TdT-negative stage suggests that it may represent a more reliable T-cell marker detectable in paraffin-embedded specimens in lymphomas of early precursors of T cells. Anti-LAT antibody showed a high sensitivity among mature T-cell and NK/T-cell neoplasms, identifying 96.3% of all cases. Two T-cell lymphomas were LAT-negative; one of them showed loss of CD3 and CD5 as well as CD43 and TCR-ß. The other, in contrast, was a NK/T-cell lymphoma strongly expressing cytoplasmic CD3. Interestingly, the three CD3-negative T-cell lymphomas that expressed LAT were positive for TCR-ß, but two of them lacked CD5. Discordant expression of TCR-ß and CD3 is not uncommon in T-cell lymphomas;35 together with the loss of other T-cell antigens35-37 this process has been called "aberrant antigen profile"35 and is considered to represent abnormal gene expression in these malignancies, rather than clonal expansion of a normal T-cell subset.35 The results obtained in the present study indicate that LAT may be helpful in the identification of T-cell lymphomas that display antigenic loss. In addition, although both LAT and CD3 were similarly high sensitive in the staining of T-cell lymphomas (96.8% and 93.6%, respectively), when used in conjunction, they successfully identified a higher number of cases (98.4%). The use of CD3 and LAT together may represent the optimal method of immunohistochemical diagnosis of T-cell neoplasms in paraffin sections.
In contrast with other T-cell lymphomas, in ALCL T-cell type, anti-CD3 labeled a higher number of cases (19/25) than anti-LAT (10/25); however, seven cases showed the LAT+CD3- phenotype, and, the use of both markers had the highest sensitivity in the identification of T-cell ALCL (22/25). Because loss of T-cell antigens is frequently found in T-cell-type ALCL,38 our results indicate that LAT is definitely of value in the phenotypic study of ALCL. Interestingly, a discordant expression of the two antigens was more frequently observed in ALCL than in other peripheral T-cell lymphomas (48.0% versus 7.2%, respectively) and this difference was statistically significant. This observation might reflect a more severe phenotypic aberration observed in these tumors.38,39 Furthermore, we also correlated the expression of LAT in ALCL with their positivity for ALK-1, and found that the majority of ALK1-positive cases (6/10) were LAT-positive, unlike ALK1-negative cases (8/32). Whether these differences indicate a functional relationship between LAT and NPM/ALK remains unknown. In addition, further clinicopathological studies are necessary to establish whether LAT expression in ALK-positive ALCL may define a subset of tumors and correlate with prognosis.12,40,41
In contrast to CD3, currently the most specific T-cell marker for paraffin sections,30,33,42 LAT shows reactivity with other hematopoietic cell types, in both normal and neoplastic conditions. LAT stained mast cells and labeled 4/5 cases of mastocytosis. It should be noted that the pattern of reactivity in mast cells differs from that shown by T lymphocytes, making distinction between these cell types easier. Finally, LAT was strongly expressed in the cytoplasm of megakaryocytes, both in normal bone marrow and in various hematological neoplasms characterized by the occurrence of atypical forms of megakaryocytes and megakaryoblasts. Because few paraffin markers for megakaryocytes are currently available and some of them, such as anti-CD61, react poorly in B5-fixed biopsies (personal observation), we conclude that anti-LAT has potential use for demonstrating megakaryocytic components in leukemias.
In conclusion, this study has confirmed the previous data on LAT mRNA expression in normal tissues by demonstration of LAT protein. The study adds new information on LAT distribution in T and NK cells and on its expression in platelets and megakaryocytes. The anti-LAT antibody has a high specificity and sensitivity for immunostaining T cells and T- and NK/T-cell lymphomas. From these results, it is evident that anti-LAT represents a valuable addition to the panel of immunohistochemical markers that can be used for lymphoma phenotyping in routinely processed tissues. Finally, anti-LAT is a promising reagent for the screening of patients with primary defects of T cell and/or platelet functions to identify anomalies of LAT gene expression.
Note added in proof: In keeping with our findings, after this paper was submitted for publication, two studies have provided evidence that LAT is expressed in platelets and plays a role during platelet activation.
Sarkar S: Tyrosine phosphorylation and translocation of LAT in platelets. FEBS Lett 1998, 441:357–360.
Gibbins JM, Briddon S, Shutes A, van Vugt MJ, van de Winkel JGJ, Saito
T, Watson SP: The p85 subunit of phosphatidylinositol 3-kinase
associate with the Fc receptor -chain and linker for activation of T
cells (LAT) in platelets stimulated by collagen and convulxin. J Biol
Chem 1998, 273:34437–34443.
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Acknowledgements |
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Footnotes |
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Supported in part by Associazione Italiana per la Ricerca sul Cancro (to M.C.) and by Biomed contract CT98-3007 (to F.F. and L.D.L.).
Accepted for publication January 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and the Ras pathway. Immunity 1998, 9:617-626[Medline]
1 SH2 domains. J Biol Chem 1992, 267:13610-13616 1 associate with a 36- to 38-kilodalton phosphotyrosine protein after T-cell receptor stimulation. Mol Cell Biol 1998, 14:4435-4442 1-binding phosphotyrosyl protein pp36/38. J Biol Chem 1998, 270:20177-20182 delta T cells: no evidence for general epithelial tropism. J Clin Pathol 1991, 44:1012-1017 delta T-cell receptor in normal human intestine and celiac disease. Hum Pathol 1991, 22:690-694[Medline]
receptor-initiated activation of natural killer cells. J Biol Chem 1995, 270:16415-16421 complex expressed in human natural killer cells. Eur J Immunol 1993, 23:1872-1876[Medline]
1, and other phosphoproteins with GRb2 and the -chain of the TCR. J Biol Chem 1995, 270:18428-18436 RI and other receptors. Curr Opin Immunol 1996, 8:766-772[Medline]
1 in an SLP-76-deficient T cell. Science 1998, 281:413-416This article has been cited by other articles:
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