Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue

Functional interactions between B and T lymphocytes are known to depend on the expression of co-stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter-receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1⁺, while centrocytes in the light zone were B7-2⁺, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2⁺ T cells were demonstrated in interfollicular areas. Intrafollicular CD4⁺ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4⁺ T cells as well as on T cells in T cell zones. The differential distribution of co-stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.     

Indirect demonstration of the lifetime function of human thymus

The aim of this study was to test the hypothesis that human thymus maintains its function as the site of early T cell development throughout life, but to a progressively diminishing extent. Mononuclear cell suspensions prepared from the samples of 39 human thymuses were analysed for the total number of cells per gram of thymus tissue, percentage of single marker-positive CD2, CD4 and CD8 cells, percentages of double-positive CD4 CD8 and CD2 CD8 cells, double-negative CD4 CD8 cells, absolute numbers of these cells per gram of tissue, and extent of the in vitro proliferation upon stimulation with concanavalin A (Con A), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) mitogens. The main outcome measures were flow cytometric data on thymus lymphoid cell composition (according to CD classification), expressed as percentages and numbers of cells per gram of thymus tissue. The total number of mononuclear cells expressed per gram of thymus tissue exponentially decreased with age. The slope of none of the analysed cell subpopulations differed from the slope of the line constructed for age-related decline of the total number of mononuclear cells (?0.024 on a semilogarithmic scale). The thymuses of all ages contained all analysed cell subpopulations in approximately the same proportions: percentages of these cell subpopulations did not change with age, except for all CD4⁺ (P = 0.017) and double-positive CD4⁺ CD8⁺ (P = 0.016) cells, which tended to decrease with age. The extent of proliferation of thymus cells upon stimulation with T and B cell mitogens was unrelated to age. We conclude that the thymus retains its function as the site of differentiation of T lymphocytes throughout life. With respect to the number of involved lymphoid cells, the function exponentially decreases with age.     

Thymic and peripheral apoptosis of antigen-specific T cells might cooperate in establishing self tolerance

Aside from CD4⁺CD8⁺ double-positive (DP) thymocytes, the subpopulations of T lineage cells affected by negative selection are unknown. To address whether this process occurs in more mature cell types, we have compared the responses of purified single-positive (SP) murine thymocytes and peripheral T cells to the superantigen staphylococcal enterotoxin B (SEB) utilizing as antigen-presenting cells (APC) a fibroblast cell line expressing transfected I-E^k class II molecules. Whereas ∽ 70% of SEB-reactive SP thymocytes, either CD4⁺ or CD8⁺, undergo programmed cell death (apoptosis) and, therefore, negative selection, CD4⁺ and CD8⁺ antigen-specific peripheral T cells are predominantly activated and proliferate to APC+SEB. Thus, mature thymocytes and peripheral T cells, with identical patterns and levels of expression of CD4, CD8 and T cell receptor (TCR), are programmed to elicit different responses followingTCR stimulation. Unexpectedly, however activation of peripheral T cells was preceded by deletion of a large fraction of Vβ8⁺ T lymphocytes (SEB specific). This surprising phenomenon was also observed in in vivo studies: in fact, administration of SEB to adult mice resulted in depletion of the majority of antigen-specific T cells from the peripheral lymphoid tissues analyzed (lymph nodes and spleen). This depletion is the consequence of deletion as indicated by program cell death of Vβ8⁺ T cells and is followed by proliferation of the remaining SEB-reactive T cells. Clonal elimination of peripheral T cells may represent a mechanism by which tolerance to self antigens never expressed in and/or exported to the thymus is achieved.     

Self-encounters of the third kind: lymph node stroma promotes tolerance to peripheral tissue antigens

Multiple mechanisms have evolved to maintain tolerance among CD8⁺ T cells to innocuous antigens that arise in cutaneous and mucosal tissues. In the thymus, medullary thymic epithelial cells directly present peripheral tissue antigens (PTAs) and incite the deletion of self-reactive thymocytes. Cross-presentation of PTAs by functionally immature, CD8α⁺ dendritic cells can lead to the deletion of self-reactive CD8⁺ T cells in secondary lymphoid organs. A third mechanism of deletional tolerance has recently been uncovered in which lymph node-resident stromal cells of non-hematopoietic origin present endogenously expressed PTAs to circulating CD8⁺ T cells. Emerging data suggest that lymph node stroma is a unique niche for controlling self-reactive T cells.     

Antigens expressed by myelinating glia cells induce peripheral cross‐tolerance of endogenous CD8+ T cells

Auto‐reactivity of T cells is largely prevented by central and peripheral tolerance. Nevertheless, immunization with certain self‐antigens emulsified in CFA induces autoimmunity in rodents, suggesting that tolerance to some self‐antigens is not robust. To investigate the fate of nervous system‐specific CD8⁺ T cells, which only recently came up as being important contributors for MS pathogenesis, we developed a mouse model that allows inducible expression of lymphocytic choriomeningitis virus‐derived CD8⁺ T‐cell epitopes specifically in oligodendrocytes and Schwann cells, the myelinating glia of the nervous system. These transgenic CD8⁺ T‐cell epitopes induced robust tolerance of endogenous auto‐reactive T cells, which proved thymus‐independent and was mediated by cross‐presenting bone‐marrow‐derived cells. Immunohistological staining of secondary lymphoid organs demonstrated the presence of glia‐derived antigens in DC, suggesting that peripheral tolerance of CD8⁺ T cells results from uptake and presentation by steady state DC.     

Characterization of human CD25+ CD4+ T cells in thymus,cord and adult blood

CD4⁺ CD25⁺ regulatory T cells prevent organ‐specific autoimmune diseases in various animal models. We analysed human lymphoid tissues to identify similar CD25⁺ regulatory T cells. Adult peripheral blood contained two populations of CD4⁺ T cells that expressed CD25 at different densities. The larger population (≈ 40%) expressed intermediate levels of CD25 (CD25⁺) and displayed a memory T‐cell phenotype (CD45RA^?/RO⁺, CD45RB^low, CD95⁺, CD62L^low, CD38^low). The smaller population of cells (≈ 2%) expressed very high levels of CD25 (CD25⁺⁺). In addition to the activation/memory T‐cell antigens mentioned above they also expressed intracellular CD152 (CTLA‐4) as well as enhanced levels of cell‐surface CD122, similar to the murine CD4⁺ CD25⁺ regulatory counterpart. To exclude that the CD25⁺⁺ cells had not been recently primed by external antigen we analysed cord blood and thymus. CD25⁺⁺, CD152⁺ and CD122⁺⁺ cells were present in paediatric thymus (10% of CD4⁺ CD8^? thymocytes) expressing signs of recent selection (CD69⁺) and in cord blood (5% of CD4⁺ cells) where they showed a naive phenotype. In addition, cord blood contained a small population of CD25⁺ cells (≈ 2% of CD4 T cells) that were CD152^? and CD122^low and displayed signs of activation. Together with published data that CD25⁺ CD25⁺⁺ cells from the thymus and peripheral blood are regulatory, our results suggest that regulatory CD25⁺ T cells leave the thymus in a naïve state and become activated in the periphery.     

Properties of mouse CD40: Cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies

We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45–50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5–10% of both CD4⁺ and CD8⁺ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40⁺, whereas CD40⁺ cells were not detectable in the thymus, even following collagenase digestion. Some 35–40% of lymphoid cells in the bone marrow of adult mice are CD40⁺ and virtually all of these are B220⁺, and hence of the B cell lineage: triple-color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre-B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up-regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non-mitogenic) anti-μ and anti-δ mAb, and moderately enhanced by co-stimulation with interleukin-4. Hyper-cross-linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti-CD40.     

Expression of B220 on activated T cell blasts precedes apoptosis

Superantigens are bacterial or viral products that polyclonally activate T cells bearing certain TCR β chain variable elements. For instance, Vβ8⁺ T cells proliferate in response to staphylococcal enterotoxin B (SEB) in vivo and then undergo Fas- and/or TNF-mediated apoptosis. We have recently shown that apoptotic SEB-reactive T cells express the B cell marker B220. Here we report the identification of a novel subset of CD4⁺B220⁺ T cell blasts that are the precursors of these apoptotic cells in SEB-immunized mice. Moreover, we show that the CD4^–CD8^–B220⁺ T cells that accumulate in the lymphoid organs of Fas ligand-defective gld mice stably express a form of the B220 molecule which exhibits biochemical similarities to that expressed by activated wild-type T cells, but is distinct from that displayed on the surface of B cells. Surprisingly, we also find a population of CD4⁺B220⁺ pre-apoptotic T cells in FasL-defective gld mice, arguing that these cells can be generated in a Fas-independent fashion. Collectively, our data support a general model whereby upon activation, T cells up-regulate B220 before undergoing apoptosis. When the apoptotic mechanisms are defective, T cells presumably down-regulate their coreceptor molecules but retain expression of B220 as they accumulate in lymphoid organs.     

The distribution of CD10 (NEP 24.11, CALLA) in humans and mice is similar in non-lymphoid organs but differs within the hematopoietic system: absence on murine T and B lymphoid progenitors

Prior studies in the human have implied an important function for CD10 (CALLA, neutral endopeptidase 24.11) in early lymphoid development. To examine the role of this ectoenzyme in an experimental system, a rat mAb specific for mouse CD10, termed R103, was generated. Immunohistological and flow cytometric analyses indicate that the distribution of CD10 in non-lymphoid anatomical compartments is virtually identical in human and mouse. However, CD10 expression within the hematopoietic system is strikingly different. In contrast to human spleen, lymph node and thymus, the corresponding mouse organs contain no detectable CD10⁺ cells. Mouse granulocytes, unlike human granulocytes, also lack CD10 expression. Five-color flow cytometric studies of adult bone marrow (BM) from C57BL/6 and BALB/c mice with mAb specific for CD43, B220, HSA, BP-1 and immunoglobulin M fail to detect any significant number of CD10⁺ cells at pro-B, pre-B or B cell stages. In addition, lymphoid cells in both (rIL-7) independent and rIL-7-dependent in vitro pro-B cell cultures lack CD10 expression. Consistent with this result, CD10 mRNA is not detected. Unlike the AA4.1⁺ population from day 13 and 14 fetal liver, the CD10⁺ subset is unable to reconstitute T and B lymphoid compartments in RAG-2^?/? mice. Nevertheless, mouse CD10 is readily found on BM stromal elements known to support early B lineage lymphoid development. Given the common expression of CD10 on human and mouse BM stromal elements, this enzyme may have an important function in the stromal cell-dependent phase of hematopoiesis.     

CD4+ T cells of both the naive and the memory phenotype enter rat lymph nodes and Peyer's patches via high endothelial venules: Within the tissue their migratory behavior differs

It is thought that naive T cells predominantly enter lymphoid organs such as lymph nodes (LN) and Peyer's patches (PP) via high endothelial venules (HEV), whereas memory T cells migrate mainly into non-lymphoid organs. However, direct evidence for the existence of these distinct migration pathways in vivo is incomplete, and nothing is known about their migration through the different compartments of lymphoid organs. Such knowledge would be of considerable interest for understanding T cell memory in vivo. In the present study we separated naive and memory CD4⁺ T cells from the rat thoracic duct according to the expression of the high and low molecular weight isoforms of CD45R, respectively. At various time points after injection into congenic animals, these cells were identified by quantitative immunohistology in HEV, and T and B cell areas of different LN and PP. Three major findings emerged. First, both naive and memory CD4⁺ T cells enter lymphoid organs via the HEV in comparable numbers. Second, naive and memory CD4⁺ T cells migrate into the B cell area, although in small numbers and continuously enter established germinal centers (GC) with a bias for memory CD4⁺ T cells. Third, memory CD4⁺ T cells migrate faster through the T cell area of lymphoid organs than naive CD4⁺ T cells. Thus, our study shows that memory CD4⁺ T cells are not excluded from the HEV route. In addition, “memory” might depend in part on the ability of T cells to specifically enter the B cell area and GC and to screen large quantities of lymphoid tissues in a short time.     

Importance of TLR2 in the direct response of T lymphocytes to Schistosoma mansoni antigens

The immunomodulatory effect of Schistosoma mansoni antigens has often been attributed to interaction with PRR expressed on APC. Our previous work has shown that S. mansoni‐soluble egg antigen (SEA) can induce, together with a Th2 response, TGF‐β‐dependent Foxp3 expression in naïve CD4⁺ T cells from NOD mice. We found that SEA can directly upregulate the expression of surface‐bound TGF‐β in purified CD4⁺ T cells in the absence of accessory cell interactions. In this study, we show that the C‐type lectin receptors DEC‐205 and galectin‐3 were involved in the direct interaction between S. mansoni antigens and CD4⁺ T cells. SEA was able to enhance CD4⁺ T‐cell secretion of bioactive TGF‐β in response to TLR2 ligand stimulation, in the absence of APC. We also show that TLR2 expressed on CD4⁺ T cells was important for the Foxp3 expression induced by SEA.     

GPA33 is expressed on multiple human blood cell types and distinguishes CD4+ central memory T cells with and without effector function

The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4⁺ T cell compartment. Naïve and CXCR5⁺ regulatory T cells were GPA33^high, and naïve conventional CD4⁺ T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4⁺ central memory T cell (Tcm) population. GPA33⁺ CD4⁺ Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33^– Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4⁺ T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4⁺ T cell lineage.     

Characterization of mouse CD6 with novel monoclonal antibodies which enhance the allogeneic mixed leukocyte reaction

Human CD6 is a cell surface protein expressed by thymocytes, mature T cells, a subset of B cells and certain cells of the brain. On human T cells, CD6 has been shown to act as a co-stimulatory molecule which modulates T cell receptor (TCR)-mediated T cell activation. To study further the recently identified mouse CD6 (mCD6), we generated and characterized a set of anti-mCD6 mAb. Anti-mCD6 mAb recognizing the mCD6 scavenger receptor cysteine-rich (SRCR) extracellular domains 1 and 3 were identified. mAb against SRCR domain 3, but not domain 1, inhibited the interaction of CD6 with a recently identified ligand, activated leukocyte cell adhesion molecule (ALCAM). Immunohistochemical analysis indicated that mCD6 expression was largely localized to the T cell areas of lymphoid tissue and, as previously reported in the human, CD6 was also expressed by neurons. CD6 was highly expressed on mouse T cells isolated from the spleen, lymph node and thymus as demonstrated by two-color immunofluorescence analysis. The CD4⁺ and CD8⁺ cells in these lymphoid compartments expressed similar levels of CD6. Immunoprecipitation studies showed that mouse thymocytes predominantly express a CD6 isoform of ～130 kDa, while splenocytes predominantly express a CD6 isoform of ～100 kDa. Anti-mCD6 mAb enhanced allogeneic mixed leukocyte reactions (MLR), indicating that CD6-ALCAM interactions may regulate the proliferative capacity of T cells.     

Opposing peripheral fates of tissue-restricted self antigen-specific conventional and regulatory CD4+ T cells

The development of self antigen-specific T cells is influenced by how the self antigen is expressed. Here, we created a mouse in which a model self antigen is conditionally expressed in different tissue environments. Using peptide:MHCII tetramer-based cell enrichment methods, we examined the development of corresponding endogenous self antigen-specific CD4⁺ T cell populations. While ubiquitous self antigen expression resulted in efficient deletion of self antigen-specific T cells in the thymus, some tissue-restricted expression patterns resulted in partial deletion of the population in peripheral lymphoid organs. Deletion specifically affected Foxp3⁻ conventional T cells (Tconv) with a bias towards high avidity TCR expressing cells in the case of thymic, but not peripheral deletion. In contrast, Foxp3⁺ Treg exhibited elevated frequencies with increased TCR avidity. T cells surviving deletion were functionally impaired, with Tconv cells exhibiting more impairment than Tregs. Collectively, our results illustrate how postthymic recognition of tissue-restricted self antigens results in opposing developmental fates for Tconv and Treg cell subsets.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with (1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; (2) Mococlonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and (3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: (1) the majority of cortical thymocytes are strongly Thy-1⁺ (positive), strongly T-200⁺, variable in Lyt-1 expression, and strongly Lyt-2⁺; (2) the majority of medullary thymocytes are weakly Thy-1⁺, strongly T-200⁺, strongly Lyt-1⁺, and Lyt-2^? (negative); (3) a minority of medullary cells are weakly Thy-1⁺, T-200⁺, strongly Lyt-1⁺, and strongly Lyt-2⁺; (4) a small subpopulation of subcapsular lymphoblasts is Thy-1⁺, T-200⁺, and negative for the expression of Lyt-1 and Lyt-2 antigens; (5) a small subpopulation of subcapsular lymphoblasts is only Thy-1⁺ but T-200^? and Lyt^?; and (6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

HLA-DR Class II expression on myeloid and lymphoid cells in relation to HLA-DRB1 as a genetic risk factor for visceral leishmaniasis

Genetic variation at HLA-DRB1 is a risk factor for visceral leishmaniasis (VL) caused by Leishmania donovani. The single nucleotide polymorphism rs9271252 upstream of the DRB1 gene provides a perfect tag for protective versus risk HLA-DRB1 four-digit alleles. In addition to the traditional role of the membrane-distal region of HLA class II molecules in antigen presentation and CD4 T-cell activation, the membrane-proximal region mediates ‘non-traditional’ multi-functional activation, differentiation, or death signals, including in DR-expressing T cells. To understand how HLA-DR contributes to disease pathogenesis, we examined expression at the protein level in circulating myeloid (CD14⁺, CD16⁺) and lymphoid (CD4⁺, CD8⁺, CD19⁺) cells of VL patients (pre- and post-treatment) compared with endemic healthy controls (EHC). Although DR expression is reduced in circulating myeloid cells in active disease relative to EHC and post-treatment groups, expression is enhanced on CD4⁺ DR⁺ and CD8⁺ DR⁺ T cells consistent with T-cell activation. Cells of all myeloid and lymphoid populations from active cases were refractory to stimulation of DR expression with interferon-γ (IFN-γ). In contrast, all populations except CD19⁺ B cells from healthy blood bank controls showed enhanced DR expression following IFN-γ stimulation. The rs9271252 genotype did not impact significantly on IFN-γ-activated DR expression in myeloid, B or CD8⁺ T cells, but CD4⁺ T cells from healthy individuals homozygous for the risk allele were particularly refractory to activated DR expression. Further analysis of DR expression on subsets of CD4⁺ T cells regulating VL disease could uncover additional ways in which pleiotropy at HLA DRB1 contributes to disease pathogenesis.     

A LARGE QUANTITY OF CD3− /CD19− /CD16− LYMPHOCYTES IN MALIGNANT PLEURAL EFFUSION FROM A PATIENT WITH RECURRENT CHOLANGIO CELL CARCINOMA

Tumor infiltrating lymphocytes (TILs) are candidates for adoptive cellular immunotherapy. Here we report on a patient whose TILs presented unusual lymphocyte antigens. Pleural effusions were collected from a 47-year-old man with recurrent cholangio cell carcinoma and malignant effusion. Effusion- associated lymphocytes (EALs) were separated by Ficoll-Hypaque gradient, and the EAL phenotype was determined by flow cytometry. The percentage of positive cells was determined for each lymphocyte-related differentiation antigen. The percentages of CD3⁺, CD19⁺, and CD16⁺ lymphocyte subpopulations among EALs were 20%, 7%, and 3%, respectively. Nearly 70% of EALs were CD3^? /CD19^? /CD56^? /CD16^?cells. The phenotypes of peripheral blood lymphocytes (PBLs) collected simultaneously from the patient's peripheral blood were CD3⁺ (52%), CD19⁺ (20%), and CD16⁺ (20%).When EALs were cultured in medium without pleural effusion, T cell-related antigens, but not B cell- or natural killer (NK) cell-related antigens, were newly expressed on EALs, and this expression reached a plateau after 48 h in culture. The proportions of CD3⁺, CD19⁺, and CD16⁺ cells were 69%, 7%, and 3%, respectively. However, when EALs were cultured in medium with pleural effusion, increased expression of T cell-related antigens was not observed; the proportions of CD3⁺, CD19⁺, and CD16⁺ cells were 16%, 6%, and 1%, respectively. Neither total cell numbers nor cellular viability of EALs changed significantly after in-vitro culture, suggesting that significant proliferation or death of EALs did not occur during the culture period. Co-culture of the patient's PBLs with autologous pleural effusion for 96 h did not alter the expression of lymphocyte-related antigens on the PBLs. These results indicate that expression of T cell-related antigens, but not B cell- or NK cell-related antigens, on EALs was blocked temporarily by the malignant pleural effusion. This is the first report concerning the existence of a large quantity of unclassified lymphocytes in which the T cell-related antigens were reversibly masked in the malignant pleural effusion.