Rapid Identification of T Helper and T Cytotoxic/Suppressor Lymphocytes with an Oxazine Dye

Peripheral blood lymphocytes displayed a plurality of sues and colors when exposed first to a methanolic solution of C.I. basic blue 141, then to an aqueous alkaline solution of the Same dye and Msed in a neutral HEPES buffer containing trace amounts of various salts. As confirmed with purified lymphocyte subpopula-tions obtained with a cell sorter, T helper cells (CD4) were small and their nuclei and cytoplasm stained deep blue. T cytotoxic/suppressor cells (CD 8) were larger than T helper cells, their nuclei stained pale green or blue green and their cytoplasm contained a cluster of magenta colored granules. From start to finish, the stain takes 15 min to perform. Used in the manner described, basic blue 141 holds promise as a rapid means of identifying and differentiating CD4 and CD8 cells under the ordinary light microscope without using monoclonal antibodies or fluorescence.     

Cross-reactivity between human and canine helper and suppressor T cell antigens using monoclonal antibodies RPA-T4 and HuLy-m8

The murine monoclonal antibodies RPA-T4 and HuLy-m8, specific for a framework determinant of human helper/inducer and suppressor/cytotoxic T cell antigens, cross-reacted with canine cell membrane molecules recognizing a biomolecular complex (50,000 to 55,000 daltons) similar to that described in humans. We investigated the distribution of these helper and suppressor T cell-like antigens on canine peripheral blood lymphocytes. With complement-mediated lymphocytotoxicity, 34% and 35% of the canine lymphocytes expressed the helper T cell-like antigens and the suppressor-like T cell antigens, respectively. When the lymphocytes were treated with RPA-T4 and HuLy-m8, the respective helper and suppressor function was significantly inhibited.     

Lycramine brilliant blue JL: a new stain for human megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte cytoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Lycramine Brilliant Blue Jl: A New Stain for Human Megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte ctyoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Selective staining methods for cartilage of rat fetal specimens previously treated with alizarin red S.

A convenient method for staining cartilage with several basic stains after alizarin red S staining of bone was investigated in rat fetuses. It was found that bromophenol blue was useful and effective for staining of the margin and center areas of cartilage, even in specimens stored in glycerin for over 10 years. The specimens were washed in running tap water for 1 hr, and subsequently were immersed in water or in 70% ethanol at pH 4 for 1 hr or longer. The specimens were then stained with 0.005% bromophenol blue in 40% ethanol adjusted to pH 4 for 2 hr, or with 0.001% bromophenol blue in 40% ethanol adjusted to pH 4 for 24 hr. Furthermore, the bromophenol blue stain color actually faded when the specimens were immersed in water or in 70% ethanol at pH 8. Descending order of the stain-effective action on fetal rat cartilage for the basic stains tested was bromophenol blue, aniline blue, Evans blue, methyl violet, trypan blue, and water blue.     

滤泡辅助性T细胞在特发性血小板减少性紫癜患者的变化及其意义

目的：研究滤泡辅助性T细胞（T follicular helper cells,Tfh）在免疫性血小板减少性紫癜（immune thrombocytopenic purpura,ITP）患者的表达并探讨其临床意义。方法：用流式细胞术检测20例健康人、25例ITP患者外周血CXCR5＋CD4＋T细胞占CD4＋T细胞的比例。结果：与健康对照组相比,ITP患者外周血CXCR5＋CD4＋T细胞占CD4＋T细胞的比例显著增高（P〈0.05）。结论：Tfh在ITP患者外周血比例增高,为Tfh能否为ITP的免疫调节和干预提出新的方向提供了证据。     

Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants

BackgroundThis study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids.MethodsA differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously.ResultsThe screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%.ConclusionThis study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.     

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with S?rensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.     

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.     

MLC-derived human helper factor(s) that promote B cell differentiation: induction, functional characterization, and role of Ia antigens

Utilizing a PFC assay to quantitate the polyclonal activation of human peripheral blood B lymphocytes, we have investigated the induction and functional activity of MLC-derived human helper factor(s). Our data demonstrate that highly purified responder T cells, but not B or null cells, are required for the elaboration of MLC helper factor(s) that trigger the in vitro differentiation of B lymphocytes into PFC. Helper factor can trigger B cell maturation in the absence of helper T cells, since complement- (C) mediated lysis of the small (less than 5%) fraction of T cells present in anti-F(ab)2 immunoabsorbent column purified B cell population eliminates the PWM induced, but not the helper factor-induced PFC response. Responder T cells required for helper factor production do not bear surface membrane Ia, since alpha p23,30 + C treatment of this population does not affect helper factor generation. In contrast, alpha p23,30 + C treatment of the allogeneic stimulator cell population eliminates helper factor production. Taken together, these results demonstrate that interaction between Ia-bearing stimulator cells and Ia- responder T cells is required for the production of MLC-derived helper factor. In additional experiments, we determined that alpha p23,30, in the absence of C, totally abrogates the PFC response triggered by MLC helper factors. This result suggests an important role for Ia antigens in the functional activity of preformed helper factor molecules.     

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.     

Modifcation of the French Azure C Tissue Stain

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

滤泡辅助性T 细胞在特发性血小板减少性紫癜患者的变化及其意义*

目的:研究滤泡辅助性T细胞(T follicular helper cells,Tfh)在免疫性血小板减少性紫癜(immune thrombocytopenic purpura,ITP)患者的表达并探讨其临床意义。方法:用流式细胞术检测20例健康人、25例ITP患者外周血CXCR5+CD4+T细胞占CD4+T细胞的比例。结果:与健康对照组相比,ITP患者外周血CXCR5+CD4+T细胞占CD4+T细胞的比例显著增高(P<0.05)。结论:Tfh在ITP患者外周血比例增高,为Tfh能否为ITP的免疫调节和干预提出新的方向提供了证据。     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Mouse lymph node germinal centers contain a selected subset of T cells--the helper phenotype

Cells staining for Lyt-1 are more frequent than cells staining for Lyt-2 in both primary follicles and the cuffs of secondary follicles; there is an even more striking predominance of cells bearing only Lyt-1 in germinal centers. In addition, there is an increase in the total percentage of cells bearing T cell antigens in germinal centers compared to primary follicles. These differences in phenotype and distribution of T cell populations indicate the T cells in B cell areas, and especially in germinal centers, are not randomly selected, but rather represent a specific subpopulation of T cells enriched for the helper phenotype (Lyt-1+2-), perhaps involved in the development and/or function of germinal centers.     

Immunologic hierarchy,class II MHC promiscuity,and epitope spreading of a melanoma helper peptide vaccine

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4⁺ T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8⁺ T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4⁺ and CD8⁺ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4⁺ T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3_281–295 (49 %) and tyrosinase_386–406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8⁺ T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4⁺ and CD8⁺ T cell responses against melanoma antigens. CD4⁺ T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8⁺ T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.     

An Automated Double Staining Procedure for Bone and Cartilage

Differential skeletal staining is an important part of developmental toxicologic studies. Traditionally these studies have required time-consuming differentiation of one or both stains used and careful attention to the maceration step to prevent specimen destruction. We present a fully automated protocol which does not require differentiation of either dye and incorporates a controlled maceration step which is highly reproducible. This has resulted in high quality staining that is reproducible, stable, and can be done in volume with minimal personnel time. The process involves the staining of skinned, eviscerated specimens fixed in 95% ethanol. Using an automated tissue processor, the specimen is stained in alcian blue for 24 hr, macerated in 3% potassium hydroxide for 24 hr and stained with murexide for 24 hr. The specimens are cleared and preserved in glycerol. Within three days specimens have red stained bone and blue stained cartilage. The procedure was developed using 20-day-old Sprague-Dawley rat fetuses to evaluate the feasibility of using the procedure for teratology studies involving the fetal skeleton. Evenly stained specimens can be examined within three days and stored for years without loss of staining.     

Anti-Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans lineage

Anti-Leu-3a, anti-Leu-3b, OKT4, and anti-T4 murine monoclonal antibodies react with a membrane component expressed by mature peripheral blood helper T cells and certain thymocyte subsets. Using a variety of immunologic staining techniques, we have demonstrated the reactivity of these antibodies with other cell types. Normal and neoplastic cells of monocyte/macrophage lineage bear the Ia+/Leu-6-/Leu-3+ phenotype, whereas histiocytosis X cells bear the Ia+/Leu-6+/Leu-3+ phenotype. The Ia+/Leu-6- cells of malignant histiocytosis and the Ia+/Leu-6+ epidermal Langerhans cells were variably Leu-3+. Normal monocyte/macrophage reactivity with anti-Leu-3/T4 appears to be primarily intracytoplasmic, whereas on U937 monocyte tumor cells, marked membrane reactivity is also observed. These results strongly suggest that certain cells other than helper T cells and thymocytes can express and, at least in some cases, synthesize a component previously regarded as T-lineage specific.     

Detection of herpes simplex virus infection using glucose oxidase-antiglucose oxidase immunoenzymatic stain

Herpes simplex virus (HSV) infected cells have been detected in tissue culture and human cell specimens by an immunoenzymatic staining method using the fungal enzyme glucose oxidase. Infected cells from culture or human specimens appear as dark blue, brown, or red, depending on the tetrazolium salt used in the disclosing reaction, with virtually no staining of uninfected cells. The specificity and sensitivity of this method and of the more commonly used immunoperoxidase method are comparable, but the immunoglucose oxidase method avoids the problems of nonspecific staining by the endogenous peroxidase present in mucosecretions and inflammatory cells. Staining time can be reduced up to 40% of that necessary for the unlabeled immunoperoxidase procedure without compromising the quality of staining results.