Aberrant antigenic expression in extranodal NK/T-cell lymphoma: a multi-parameter study from Thailand

Background

Extraosseous plasmacytoma, so called extramedullary plasmacytoma (EMP) is relatively rare in China. The aim was investigate the clinicopathologic features of EMP and the role of Immunophenotype and genotype detection in diagnosis of EMP.

Methods

Thirty-two cases of EMP were investigated retrospectively by histopathology, immunophenotype, genotype and survival analysis.

Results

Clinically, the mean age of the patients was 53.4. Most of the patients received no treatment after the diagnosis was established, and the prognosis was relatively poor. Histologically, in 40% of the cases, the neoplastic cells were grade II or III. The neoplastic cells expressed one or more PC associated antigens. The immunophenotype of EMP and inflammation of sinonasal regions with numerous PC infiltrations were compared and showed some difference in expression of CD45, CD27, CD44v6 and Bcl-2 as well. Ig light chain restriction was detected in 87.5% of the cases.

Conclusions

we described 32 Chinese cases of EMP, compare with that reported in the literature, some differences are presented, including higher percentage of grade II and III cases, clinically inconsistent treatment and management as well as poor outcome of the disease.     

Plasmacytoma with aberrant expression of myeloid markers, T-cell markers, and cytokeratin.

Plasmacytomas are localized neoplastic proliferations of monoclonal plasma cells. When multifocal, the process is referred to as multiple myeloma. These lesions exhibit a pattern of antigen expression and cytomorphology that usually leads to a ready diagnosis. However, potentially troublesome variations in immunophenotype occur. We describe a case of a plasmacytoma from a patient who presented with sudden onset of pain and a lytic lesion of the left proximal humerus. Hematoxylin and eosin-stained sections showed a lymphoproliferative lesion composed of large lymphoid cells, some with plasmacytoid and immunoblastic features. The lesion also showed significant mitotic activity. Immunohistochemical staining was positive for CD45 (LCA), CD56 (N-CAM), CD43 (MT1), and cytokeratin CAM5.2. There was also clonal staining for lambda light chains. In addition, flow cytometric analysis showed positivity for myeloid markers such as CD13, CD33, CD38, and CD138. Significant negative markers include CD20 (L26), CD45RO (UCHL-1), and CD79alpha. The unusual phenotypic features of this plasmacytoma illustrate potential diagnostic pitfalls. It is important to fully study such lesions to correctly classify them, because this has significant impact on prognosis and management.     

An approach to diagnosis of T-cell lymphoproliferative disorders by flow cytometry

T-cell lymphoproliferative disorders are among the most challenging diagnoses in hematopathology. Unlike the more common B-cell disorders, in which clonality is often readily discernible by surface immunoglobulin light chain restriction, there is no specific immunophenotypic signature that is diagnostic of a clonal T-cell population. Immunophenotypic criteria that are helpful in the diagnosis of T-cell neoplasms include T-cell subset antigen restriction, anomalous T-cell subset antigen expression, deletion or diminution of one of the pan T-cell antigens, a precursor T-cell phenotype, and expression of additional markers (e.g., CD30, CD20, major myeloid antigens, and TCRgammadelta). Analysis of the inherent forward and orthogonal light scatter properties of the cell can also provide important diagnostic clues. None of these features is 100% specific, however, for aberrant expression of pan-T antigens may be seen in viral infections, B-cell malignancies, or in reactive changes following administration of certain medications. An increased CD4:CD8 ratio is often observed in Hodgkin's lymphoma. Based on the analysis of 87 neoplastic and 80 control cases, we conclude that flow cytometric features that are most suspicious for malignancy include the loss or markedly dim expression of CD45; complete loss of one or more pan-T antigens; diminished expression of more than two pan-T antigens in conjunction with altered light scatter properties; and CD4/CD8 dual-positive or dual-negative expression (except thymic lesions).     

Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex.

Reproducible visualization of neurons and glia in human brain is essential for quantitative studies of the cellular changes in neurological disease. However, immunohistochemistry in human brain specimens is often compromised because of prolonged fixation. To select cell lineage-specific antibodies for quantitative studies of neurons and the major types of glia, we used 29 different antibodies, different epitope retrieval methods, and different detection systems to stain tissue arrays of formalin-fixed human brain. The screening pointed at CD45/leukocyte common antigen (LCA), CD68(KP1), 2',3' cyclic nucleotide phosphatase (CNPase), glial fibrillary acidic protein (GFAP), HLA-DR, Ki67, neuronal nuclei (NeuN), p25alpha-antigen, and S100beta as candidates for future cell counting purposes, because these markers visualized specific neuronal and glial cell bodies. However, significant negative correlation between staining result and formalin fixation was observed by blinded scoring of staining for CD45/LCA, CNPase, GFAP, and NeuN in brain specimens fixed by immersion and stored up to 10 years in 4% formalin solution at room temperature, independent of donor sex and postmortem interval. In contrast, improved preservation of NeuN and CNPase staining, and full preservation of GFAP and CD45/LCA staining in tissue fixed by perfusion and stored for up to 3 years in 0.1% paraformaldehyde solution at 4C, indicated that immunohistochemistry can be performed in well-preserved biobank material.     

The functional heterogeneity of CD8+ cells defined by anti-CD45RA (2H4) and anti-CD29 (4B4) antibodies.

The monoclonal antibodies, anti-2H4(CD45RA), and anti-4B4(CD29), along with UCHL1-(CD45RO), identify reciprocal populations of CD4 cells with distinct suppressor inducer (CD45RA+CD29-CD45RO-) and helper inducer (CD45RA-CD29+CD45RO+) functions. Although the CD8+ population is known to contain precytotoxic, cytotoxic, suppressor, and some natural killer cells, the exact phenotypic identities of these functional CD8 subsets has not been established. In this study, we tried to determine whether these monoclonal antibodies could distinguish functionally distinct subsets of cells within the CD8+ population. For this purpose, whole T cells or fractionated T cells were sensitized with irradiated allogeneic non-T cells for 6 days, following which, CD8+ or CD8+CD11b- cells were isolated and cellular functions such as suppressor, killer precursor, and killer effector activity were assessed. The results showed that both class I-restricted alloantigen-specific killer effector and killer precursor cells belonged to the CD8+CD11b-CD45RA-CD29+ population. Moreover, these killer effector cells expressed the CTL-associated S6F1 molecule, an epitope of the LFA-1 antigen. In contrast, suppressor effector cells belonged to the CD8+CD11b-CD45RA+CD29- cell population. Although the UCHL1 antigen has been reported to define the CD4+CD29+ helper inducer cell, over 90% of allo-activated CD8+ cells expressed this antigen, whereas only 40-60% of these cells expressed either CD45RA or CD29 antigens. These results suggest that anti-CD45RA and anti-CD29 antibodies may provide useful tools for distinguishing between suppressor effector versus killer effector and killer precursor cells within the CD8+CD11b- population.     

Sera from patients with multiple sclerosis react with human cell T lymphotropic virus-I gag proteins but not env proteins--Western blotting analysis

To study the possible involvement of human T cell lymphotropic virus type I (HTLV-I)-related agent in Japanese multiple sclerosis (MS), we performed a Western blotting analysis, using purified viral antigens, on sera from 46 patients with MS, nine patients with other neurologic diseases, and 11 healthy controls. Of 46 MS patients, 11 (24%) had antibodies reactive with antigens corresponding to the group-specific antigen (gag) proteins (p15, p19, and p24), although the prevalence was lower than that reported in a recent study using an enzyme-linked immunosorbent assay (ELISA). Despite the lower frequency of immunoreactivity, Western blotting technique had merits of identification of multiple antigens and higher specificity for detection of antibodies than ELISA. Those sero-positive patients consisted of four cases with IgG antibodies reactive mainly to the gag p24 and/or p15, four with IgM antibodies mainly to the gag p24 and/or p19, and three with both IgG and IgM antibodies. These immunostaining patterns of MS sera were clearly distinguishable from those of adult T cell leukemia patients who had antibodies to the envelope (env) proteins and its precursors in addition to the gag proteins. The antibody in MS sera was generally of low titer and reactive at a high serum concentration (1/10 dilution). None of the sera from patients with other neurologic diseases and healthy controls had the viral antibodies. These findings indicate that at least one quarter of Japanese MS patients have antibody responses to a hitherto unidentified agent related to HTLV-I, which possibly plays a part, primarily or secondarily, in the pathogenesis of those patients.     

Carbohydrate and peptide antigens in macrophage populations derived from human bone marrow and milk: an immunomorphological and immunochemical analysis

Summary An immunomorphological and immunochemical study was performed to elucidate the pattern of carbohydrate antigens and their relationships to the cluster differentiation (CD) 68 epitopes on macrophages derived from human bone marrow and milk. Core and backbone antigens recognized by lectins fromBauhinia purpurea (BPA),Helix pomatia (HPA),Arachis hypogaea (PNA),Glycine max. (SBA),Griffonia simplicifolia (GSA-I-B₄),Lycopersicon esculentum (LEA) andErythrina cristagalli (ECA) were expressed by both macrophage populations. Additionally, they exhibited various peripheral type 1 and type 2 carbohydrate antigens. In bone marrow trephine biopsies, the number of macrophages stained by the CD68-specific monoclonal antibody PG-M1 exceeded significantly (range 30–40%) the subpopulation expressing SBA, GSA-I-B₄ and ECA binding sites as well as the Lewis^a antigen. This result is very interesting since, fromin vitro studies, GSA-I-B₄ and SBA are known to react especially with activated macrophages. Western blotting experiments on milk macrophage lysates revealed that ECA, GSA-I-B₄, BPA, PNA and MAA visualize a 110 kDa band isographic with the CD68 antigen detected by PG-M1, KP1 and Ki-M1P monoclonal antibodies. These antibodies recognize peptide epitopes as shown by enzyme-linked immunosorbent assays after biochemical modification of milk macrophage lysates. This result is in keeping with the assumption that the CD68 antigen consists of a highly glycosylated mucin-type glycoprotein comprising various differentiation-dependent epitopes.     

In vitro stimulation of peripheral blood mononuclear cells by phytohaemagglutinin A induces CD45RA expression on monocytes.

Following phytohaemagglutinin A stimulation, CD45RA positive monocytes increased from 12.2 +/- 8.9% to 63.5 +/- 8.8% (p < 0.001), without a significant change in cell surface antigen density. In contrast, HLA-DQ antigen, also expressed in 76.8 +/- 10.5% of the monocytes after PHA stimulation for 48 h, revealed a marked enhancement in fluorescence intensity (p < 0.001). This up-regulation is already evident in unstimulated cultures. The percentages of CD45RA and HLA-DQ positive monocytes were correlated (r = 0.80, p < 0.001), substantiating a previous clinical observation. CD45RA expression may probe activated mono/macrophages.     

Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits

Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections.     

Split T cell tolerance against a self/tumor antigen: spontaneous CD4+ but not CD8+ T cell responses against p53 in cancer patients and healthy donors

Analyses of NY-ESO-1-specific spontaneous immune responses in cancer patients revealed that antibody and both CD4(+) and CD8(+) T cell responses were induced together in cancer patients. To explore whether such integrated immune responses are also spontaneously induced for other tumor antigens, we have evaluated antibody and T cell responses against self/tumor antigen p53 in ovarian cancer patients and healthy individuals. We found that 21% (64/298) of ovarian cancer patients but no healthy donors showed specific IgG responses against wild-type p53 protein. While none of 12 patients with high titer p53 antibody showed spontaneous p53-specific CD8(+) T cell responses following a single in vitro sensitization, significant p53-specific IFN-γ producing CD4(+) T cells were detected in 6 patients. Surprisingly, similar levels of p53-specific CD4(+) T cells but not CD8(+) T cells were also detected in 5/10 seronegative cancer patients and 9/12 healthy donors. Importantly, p53-specific CD4(+) T cells in healthy donors originated from a CD45RA(-) antigen-experienced T cell population and recognized naturally processed wild-type p53 protein. These results raise the possibility that p53-specific CD4(+) T cells reflect abnormalities in p53 occurring in normal individuals and that they may play a role in processes of immunosurveillance or immunoregulation of p53-related neoplastic events.     

Flow cytometric immunophenotypic characteristics of plasma cell leukemia

The aim of this prospective study was to define the flow cytometric characteristics of simultaneously investigated bone marrow and peripheral blood plasma cells antigens expression in 36 plasma cell leukemia (PCL) patients. The immunophenotypic profile of plasma cells was determined with a panel of monoclonal antibodies. The antigen expression intensity was calculated as relative fluorescence intensity (RFI). Bone marrow plasma cells showed expression of particular antigens in the following proportion of cases: CD49d 100%, CD29 94%, CD54 93%, CD44 83%, CD56 60%, CD18 26%, CD11b 29%, CD11a 19%, CD117 27%, CD71 30%, CD126 100% and CD19 0%, while the expression of those antigens on peripheral blood plasma cells was present in the following percentage of patients: CD49d 100%, CD29 96%, CD54 93%, CD44 95%, CD56 56%, CD18 50%, CD11b 53%, CD11a 29%, CD117 26%, CD71 28%, CD126 100% and CD19 0%. The expression of CD54 was significantly higher than that of adhesion molecules belonging to the integrin b2 family: CD11a, CD18 and CD11b, on both bone marrow and peripheral blood cells (p < 0.01). Expression of CD18, CD11a and CD11b was differential between two cell compartments: lower on bone marrow and higher on peripheral blood cells. We found that plasma cells in the bone marrow of patients with plasma cell leukaemia showed significantly greater granularity and size than those in the peripheral blood (p = 0.0001 and p = 0.04, respectively). However, no differences in cell size or granularity were revealed between bone marrow plasma cells from patients with PCL and multiple myeloma. In conclusion, impaired expression of adhesion molecules such as CD11a/CD18 (LFA-1) or CD56 may explain hematogenic dissemination characterizing PCL. The following pattern of adhesion molecule expression according to the proportion of plasma cells expressing a given antigen in peripheral blood and bone marrow and arranged in diminishing order may be established: CD49d > CD44 > CD54 > CD29 > CD56 > CD18 > CD11b > CD11a. Immuno-phenotyping of plasma cells in PCL, as in multiple myeloma, might be useful in detecting minimal residual disease in cases with aberrant antigen expression and for selecting therapeutic agents towards specific membrane targets.     

The subpopulation structure of human T-lymphocytes studied by using a monoclonal antibody (MCA) to CD27]

The obtained murine mAb LT27 (IgG2a) assigned to the cluster of differentiation CD27 was used to study the distribution of antigen CD27 among human lymphocytes scbpopulations in normal state and immunopathology. In normal donors the antigen CD27 was found to be expressed most frequently on CD4+ cells (90 +/- 8% of which coexpressed antigen CD27) and to the lesser extent on- CD8+ cells (only 77 +/- 28% of CD8+ cells carried antigen CD27). 79 +/- 12% of double negative lymphocytes (CD3+CD4-CD8-) expressed antigen CD27. In patients with hypogammaglobulinemia the proportion of CD4+CD27+/CD4+ and CD8+CD27+/CD8+ was significantly reduced to 80 +/- 11% (p < 0.01) and 45 +/- 19% (p < 0.001), respectively. The ratio CD4+CD27+/CD4+ varied insignificantly with the increase of CD4+ population, but the increase of the CD8+ population was accompanied by the definite tendency to a decrease of the ratio CD8+CD27+/CD8+. The distribution of CD27 antigen inside CD4+ and CD8+ subpopulations was found to be different from the distribution of CD29 and CD45RA antigens.     

Characterization of Vitiligo Antigens

Patients with vitiligo have circulating antibodies directed in part to pigment cell antigens with MWs of approximately 90, 75, and 40-45 kDs. These antigens are denominated VIT 90, VIT 75, and VIT 40, respectively. To further characterize these “vitiligo” antigens, we examined their relation to antigens defined by a panel of 25 monoclonal antibodies (moab) to pigment cell antigens. We found by immunoprecipitation and SDS-PAGE analysis of ¹²⁵I labelled, detergent soluble, human melanocyte macromolecules, that 24 (83%) of 29 patients with vitiligo had antibodies to one or more vitiligo antigens vs. 2 (7%) of 28 control individuals. Seventeen of the 25 moabs did not react with any labelled antigen in the same lysate. Of the remaining eight moabs, only four precipitated an antigen that co-migrated with one of the vitiligo antigens. Moab TA99, HMSA-5, and TMH-1 (all directed to the 75 kD tyrosinase-related protein [TRP1]) co-migrated with VIT 75. Moab W6/32 (directed to class I HLA antigen) co-migrated with VIT 40. Immunodepletion studies with vitiligo antibodies selectively depleted the antigen defined by W6/32 but not the antigen defined by TA99 and HMSA-5, indicating that VIT 75 was not the 75 kD tyrosinase-related protein. The vitiligo antigens were easily labelled by the lactoperoxidase technique but poorly labelled with ³⁵S-methionine, suggesting they are expressed on the cell surface. These studies indicate that VIT 90 and VIT 75 differ from antigens defined by currently available moabs to pigment cell antigens. VIT 40 appears to share a cross-reactive epitope, or be tightly bound to, class I HLA antigen.     

CD45RB Is a Novel Molecular Therapeutic Target to Inhibit Aβ Peptide-Induced Microglial MAPK Activation

Background

Microglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer''s disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed β-amyloid (Aβ) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation.

Methodology and Results

In this study, CD45RB modulation of Aβ peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with “aged” FITC-Aβ_1–42 and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Aβ_1–42 peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-α and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Aβ_1–42 peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Aβ_1–42 peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-α and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Aβ_1–42 peptide, inhibits co-localization of microglial MHC class II and Aβ peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells.

Conclusion

In summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Aβ phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Aβ lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.     

Adhesion-mediating molecules of human monocytes

Adhesion of monocytes to each other and to T cells and substrates is increased by phorbol esters. In the presence of these compounds monocyte aggregation was almost completely inhibited (greater than 90%) by monoclonal antibody 60.3. This antibody recognizes GP90 (CD18), a leukocyte surface glycoprotein which is separately and noncovalently associated to either GP160 (CD11a), GP155 (CD11b), or GP130 (CD11c). Anti-LFA-1 antibody (CD11a) was only partially inhibitory (35%) while antibodies 60.1 (CD11b) and anti-Leu-M5 (CD11c) had a minimal inhibitory effect (10%). Antibody LB-2 recognizing a single glycoprotein distinct from the GP90-GP160 complex and expressed on activated B and T cells, monocytes, and vascular endothelial cells was partially inhibitory (22%). Monoclonal antibodies anti-C3bR (CD35), T29/33 (CD45, leukocyte common antigen 200). TA-1 (CD11a), OKM1 (CD11b), F10-44-2 (brain-leukocyte antigen), OKM5 (monocyte-endothelial cell antigen) and to class I or class II molecules exerted no inhibition on the monocyte aggregation. Fab fragments of antibody 60.3 efficiently inhibited not only monocyte aggregation in the absence or presence of phorbol esters but also adhesion of these cells to autologous or allogeneic T lymphocytes and, to a lesser extent, to plastic surfaces. It is thus concluded that GP90, either alone or associated to the larger glycoproteins, and LB-2 antigen mediate monocyte adhesion.     

Immune response to individual maedi-visna virus gag antigens

The lesions caused by maedi-visna virus (MVV) are known to be immune mediated with a presumed contribution by the response to viral antigens. However, very little is known about the T-cell response to individual viral proteins. We have therefore expressed the three individual gag antigens of MVV strain EV1 (p16, p25, and p14) in a bacterial expression system and used the purified recombinant proteins to analyze the antibody and CD4+ T-cell response to MVV. Plasma samples were taken from sheep after 1 year of infection with MVV. The titers for antibodies in these samples were determined by indirect enzyme-linked immunosorbent assays and were as follows: anti-p25 antibody, 1:400 to >1:3,200; anti-p16 antibody, 1:400 to 1:3,200; and anti-p14 antibody, 1:<100 to 1:3,200. When the induction of antibodies was followed over time postinfection (p.i.), samples positive for anti-p25 were seen by day 24 p.i., followed by anti-p16 by day 45 p.i., and lastly anti-p14 by day 100 p.i. T-cell proliferative responses to all three gag antigens were detected in persistently infected sheep peripheral blood lymphocytes. The antigens were therefore used to raise T-cell lines from persistently infected sheep. These T-cell lines were shown to be specific for the recombinant gag antigens and for viral antigen expressed on infected macrophages. The proliferative response was restricted to major histocompatibility complex class II HLA-DR and so was due to CD4+ T lymphocytes. All three gag antigens may therefore play a role in immune-mediated lesion formation in MVV disease by presentation on infected macrophages in lesions.     

Effect of dextran-S (alpha, 1-3 dextran) on the growth of plasmacytomas MOPC-104E and J558

The murine plasmacytomas MOPC-104E and J558 secrete IgM-lambda and IgA-lambda, respectively, which are antibodies to alpha, 1-3 dextran, a constituent of dextran-S (DEX-S) extracted from Leuconostoc mesenteroides. A single i.p. injection of 10 microgram DEX-S into BALB/c mice from 7 days before and up to 3 days after the implantation of 5 x 10(3) plasmacytoma cells protected the BALB/c mice from developing the tumors. Immunization with other antigens, such as Escherichia coli lipopolysaccharide, inulin (alpha, 1-6 linkage), and dextran T-10 (no known alpha, 1-3 linkages) did not protect the mice from developing the tumors. Growth of LPC-1 was not affected by DEX-S. The mechanism of this growth inhibition is unknown. It does not appear to depend solely on binding of antigen to tumor cells, and the limits of the effective time intervals between antigen and tumor injection suggest dependence on the presence of antibody to alpha-1,3 dextran.     

Immunoregulatory T lymphocytes in man. Soluble antigen-specific suppressor-inducer T lymphocytes are derived from the CD4+CD45R-p80+ subpopulation

Regulation of the immune response in man is largely dependent on interactions between cells of the cluster designation 4+ (CD4+) helper/inducer sublineage and the CD8+ suppressor/cytotoxic sublineage. When cultured with autologous antigen-primed CD4+ lymphocytes, CD8+ cells differentiate into suppressor T cells (Ts) that specifically inhibit the response of fresh autologous CD4+ cells to the priming antigen only. The current study was undertaken to analyze the roles in this suppressor circuit of subpopulations of the CD4+ sublineage distinguished from one another on the basis of their binding (or lack of binding) to monoclonal antibodies against molecules p80 (Leu8) and CD45R (p220/Leu18/2H4). When examined for the proliferative responses to alloantigenic stimuli, each of the four: CD4+p80+, CD4+p80-, CD4+CD45R+, and CD4+CD45R- populations proliferated vigorously, synthesized interleukin 2 (IL-2) and interferon and released soluble IL-2 receptors. However, the responses to soluble antigens such as Candida and diphtheria toxoid were exhibited by CD4+CD45R-, CD4+p80+, and CD4+p80- cells, but not by CD4+CD45R+ cells. When examined for their ability to induced CD8+ Ts in the Candida-driven suppressor-induction culture system, only CD4+p80+ and CD4+CD45R- cells induced strong suppression. Further, when CD4+CD45R- cells were separated into CD4+CD45R-p80+ and CD4+CD45R-p80- subpopulations, despite the ability of both subpopulations to respond to Candida, only CD4+CD45R-p80+ cells induced autologous CD8+ Ts. Activated CD8+ Ts suppressed not only proliferation but also the release of soluble IL-2 receptors by autologous antigen-activated CD4+ cells. Thus, the antigen-specific suppressor-inducer T cells appear to be derived from the CD4+CD45R-p80+ (Leu3+, Leu8+, 2H4-) subpopulation of the CD4+ sublineage.     

Adhesion characteristics of human interleukin 2-activated natural killer cells

Culture of human monocyte-depleted peripheral blood mononuclear cells with recombinant IL2 (rIL2) induced adherence to plastic by 24 hr and subsequent proliferation in a subpopulation of lymphocytes with phenotypic and functional characteristics of activated natural killer (NK) cells. Purified human NK cells activated in the presence of IL2 for 24 hr upregulated the expression of the CD11c (p150.95) and CD11a antigen but not other cellular adhesion molecules (CAM). After further incubation with IL2, NK cells displayed upregulation of all of the antigens in the CD11/CD18 family of CAM. The process of adhesion was strictly dependent on culture in the presence of IL2, divalent cations, and active cellular metabolism. Adhesion also was dependent on expression of CAM on the cell surface, since monoclonal antibodies to CAM inhibited adhesion of activated NK cells to varying degrees (from 50 to 80%). An antibody (LeuM5) to the CD11c antigen (p150.95) gave the highest level of inhibition, and anti-CD11a (LFA-1) also was inhibitory, while anti-CD56 (NKH1) or anti-CD11b did not interfere with adhesion to plastic. Anti-CD11c was also the most effective in initiating the detachment of adherent-phase NK cells. Antibodies to CD18 or CD2 antigen also inhibited binding of NK cells to plastic. The blocking effects of anti-CD2 and anti-CD11a were additive in this system. On the surface of plastic-adherent and motile NK cells, all CAM except the CD56 antigen had a polar or bipolar distribution, as determined by staining with anti-CAM antibodies. Surface antigens CD11b, CD11c, CD2, and CD18 on nonadherent NK cells were clustered at the cellular poles by both immunofluorescence and immunogold electron microscopy, whereas CD11a (LFA-1) and CD56 antigens were distributed diffusely. CAM, especially CD11c, were also detected in cytoplasmic granules by immunostaining in IL2-activated NK cells. Thus, CAM may be stored in granules, allowing for their rapid transfer to the cell membrane in response to activation. Our results indicate that CAM are upregulated in IL2-activated NK cells and that some of these molecules (e.g., CD11c) play an important role in the development of plastic adherence by a subpopulation of these cells.     

Immunological and phenotypic characterization of cell constituents of breast milk

The morphology and immunological phenotype of plastic-adherent cells from human colostrum were studied in an in vitro short-term culture (6–7 days) using antibodies to CD3, CD31, CD34, CD45, CD68, vimentin, and osteocalcin. In 20% of the analyzed cultural flasks, nearly equal proportion of fibroblast-like cells and rounded cells was observed. In the 80% of the flasks, cells of regular shape were detected with the presence of single fibroblasts. The diameter of flattened cells ranged from 10 to 100 μm. All plastic-adherent cells did not express CD3 and showed weak binding (w) to the antibodies against CD31, CD34, and CD45. At the same time, we identified adherent cells that readily bound the antibodies against CD68, vimentin, and osteocalcin. According to the literature data, the CD68 + CD3^–CD31^wCD34^wCD45^w immunological phenotype of the majority of the adherent cells from the colostrum allows them to be classified as monocytes/macrophages. High expression of stromal antigens—vimentin and osteocalcin—in 40–45% of the adherent cells in the culturing medium lacking any osteogenic supplements (β-glycerophosphate, ascorbic acid, and dexamethason) implies the presence of osteoblasts in the colostrum, which differentiate from mesenchymal stem cells under the action of humoral factors contained in breast milk.