Immunohistological identification of the activated lymphocytes in human palatine tonsil

Employing various monoclonal antibodies and immunoperoxidase technique, we studied the distribution of the activated lymphocytes in human tonsillar tissue. Both the activated T-cells positive for IL2-receptor (IL2-R) and the activated B-cells defined by L29 were seen in the interfollicular area. Vast majority of the lymphocytes in the germinal center was stained with the L29, the anti-transferrin-receptor antibody, and the Ki-67 antibody which reacts with cycling cells in G1, S, or G2 + M phase. On the other hand, scarcely any cells in the mantle zone were stained with those. The Ki-67 positive cells in the germinal center were identified as two types of staining pattern, i.e., with strong nucleolar staining found in the dark zone and with nuclear staining found in the light zone. Small number of IL2-R positive cells was found in the mantle zone. The cell number per unit area of the activated lymphocytes in the interfollicular area as well as in the germinal center was decreased as the increment of patients' age. From these results, the immunological activation system of T- and B-cells in the tonsils was discussed.     

Characterization of lymphocyte subpopulations in warthin's tumor

Cell suspensions from six Warthin's tumors (WTs) were characterized with fluorescence-labeled cell cytometry. WT lymphocyte subsets were identified with monoclonal antibodies directed against lymphocyte-associated cell antigens including T lymphocyte subsets, B lymphocytes, and natural killer (NK) cells. Results showed that T cell proportions were 58% and B cell proportions were 39%. The T cell helper:cytotoxic-suppressor ratio was 5.7:1 and the B to T cell ratio was 0.8:1. NK cells represented 1.3% of cells. When compared to peripheral blood lymphocytes (PBLs) in the same patients, statistically significant differences were noted between PBLs and WT lymphocytes in the percentage of B lymphocytes (P<.01), T cytotoxic-suppressor lymphocytes (P<.02), NK cells (P<.01), and in the ratios of B to T lymphocytes (P<.01) and T helper to T cytotoxic-suppressor lymphocytes (P<.03). Comparing these data to retrospective data on lymphocyte distribution in normal and reactive lymph nodes, the epithelial component does not appear to exert a local effect on the lymphoid component of WT.     

Cytokines locally produced by lymphocytes removed from the hypertrophic nasopharyngeal and palatine tonsils

OBJECTIVE: Human palatine tonsils and the nasopharyngheal tonsil are the largest components of the Waldeyer's ring. Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils are responsible for the local and the systemic immune response. We studied the cytokine production by lymphoid cells isolated from 16 nasopharyngeal tonsils (adenoid) and 9 palatine tonsils surgically removed by from 25 children (aged from 4 to 15 years) suffering from tonsil hypertrophy. METHODS: We evaluated (by the cytometry method, using BD Bioscience kits, San Diego, CA) the concentration of IL-2, IL-4, IL-5, IL-10, TNF(alpha) and IFN(gamma) released from human peripheral blood mononuclear cells (MC) (activated or not activated by phytohaemagglutinin (PHA)) cultured in vitro during 72 h. The fluorescence-activated cell sorter (FACS) analysis was also performed and the percentage of mononuclear cells (unstimulated or activated by phorbol acetate during 24 h) stained with the monoclonal antibodies anti-CD3 containing the intracellular cytokines was calculated. RESULTS: The increased secretion of IL-2, IL-4, IL-5, TNF(alpha) and IFN(gamma) from PHA activated palatine origin immune cell cultures, as compared to adenoids, was revealed. The higher mobilization (Delta%) of CD3+ T-lymphocytes containing IL-12 in palatine cell cultures (798.5+/-276.29%), in comparison with to the adenoids (298.5+/-49.16%; p< or =0.05), was also noted. CONCLUSION: In palatine tonsils, as compared to adenoids, the cellular immune (Th1) response dominates over humoral immune (Th2) reaction.     

Immunologically activated cells in aural cholesteatoma.

In this immunohistochemical study, we characterized the cells infiltrating the stroma of acquired aural cholesteatomas in detail, using a panel of monoclonal antibodies directed against immune cell type-specific antigens, HLA class II antigens, and interleukin-2 receptor. For all antibodies used, normal ear skin was stained for comparison. The vast majority of the infiltrating cells was CD45-positive, ie, derived from bone marrow. Reactivity with anti-CD3 and anti-CD6 antibodies revealed an abundant infiltration of T lymphocytes beneath the squamous epithelium of cholesteatoma. The B lymphocyte-specific anti-CD19 and anti-CD22 antibodies detected only occasional positive cells. Hence, the cellular infiltrate in the stroma of aural cholesteatoma is made up primarily of T cells with macrophages scattered between them. Expression of HLA-DR was almost as high as that of CD45, whereas CD25-positive cells were detected in lower amounts. We infer that the majority of T cells and macrophages in the stroma of cholesteatoma are in an immunologically activated state. The characteristics of the infiltrating cell population suggest an antigen-driven process in cholesteatoma.     

Histologic and immunohistochemical characterization of tumor and inflammatory infiltrates in oral squamous cell carcinomas treated with local multikine immunotherapy: the macrophage at the front line

Squamous cell carcinomas of the head and neck (SCCHN) are excellent candidates for local immunotherapy owing to their accessibility and their infiltration by mononuclear cells that are susceptible to immunomodulation. A response rate of 25–60% has been reported for treatment with natural IL-2 or a mixture of natural lymphokines. In the present study, biopsies and posttreatment excision specimens from nine patients with operable SCCHN treated systemically with a variety of immunomodulators and locally with natural lymphokines (multikine, CelSci) were analyzed in an attempt to correlate clinical response to histopathological and immunohistochemical changes. Formalin-fixed, paraffin-embedded tissues were stained with antibodies against lymphocytes (CD45, CD3, CD4, CD8, CD20), macrophages (CD68) including dendritic cells (S-100), markers for lymphocyte activation (CD30, HLA-DR), natural killer cells (CD56 and CD57), beta-2-microglobulin and keratin. One patient showed a complete response to treatment and two a partial response. Tumor size was significantly smaller after therapy. Clinical and pathological regression were more prominent in the smaller tumors. Numerous macrophages, both mononucleated and multinucleated, were present along the tumor-stroma interface in the posttreatment specimens of seven patients, most prominently in the three patients with tumor regression. The increase in the number of CD68+ and S-100+ macrophages after treatment was statistically significant. Lymphocytic infiltrates, which showed some increase following treatment, were composed of a mixture of T and B lymphocytes, the former mostly in contact with the tumor and the latter placed more peripherally. CD8+ lymphocytes extended into the tumors, whereas CD4+ lymphocytes showed minimal extension. Intensity of beta-2-microglobulin staining in tumors was significantly higher following therapy and associated with a better outcome. The marked increase in macrophages following treatment may indicate that the macrophage plays a major role in tumor recognition, destruction and clearance. An increase in the number of macrophages in a posttreatment specimen may indicate immunoresponsiveness.This study is a CISEPO project and was supported in part by the Saul A. Silverman Family Foundation.     

An immunohistologic study of the distribution and status of activation of head and neck tumor infiltrating leukocytes

Summary We examined tumor infiltrating leukocytes (TIL) in frozen sections of 28 biopsies from squamous cell carcinomas of the head and neck (SCCHN). In so doing, we used monoclonal antibodies (MoAb) directed against various leukocyte antigens. As defined by HLe-1+ cells, leukocyte infiltration was present in all biopsies. The amount of HLe-1+ cells was more often greater in stage III than in stage IV lesions. Most of the TIL were identified as CD5+ T-lymphocytes. In contrast, CD19+ B-cells were sparse in most biopsies. CD14+ monocytes/ macrophages were found in only a few specimens. The relative proportion of CD4+ T-helper cells was higher than or at least equal to CD8+ suppressor/ cytotoxic cells in all samples tested. Interleukin-2 (IL-2) receptor+ lymphocytes were evident in 13 of 22 biopsies stained for CD25 reactivity, and were more often observed in stage III than in stage IV tumors. All biopsies from recurrent tumors had no detectable IL-2 receptor+ cells.Our findings provide evidence for a positive correlation between a greater amount of TIL in earlier stages of SCCHN. The presence of IL-2+ lymphocytes suggests that SCCHN may be capable of activating resting lymphocytes for further IL-2¡nduced proliferation.     

Evaluation of cytochrome concentration in peripheral blood lymphocytes of patients with laryngeal cancer

The aim of the study was evaluation of cytochrome concentration in the peripheral blood lymphocytes of patients with laryngeal cancer. The study was conducted in a group of 62 patients presenting different clinical advancement of the disease. The study material consisted of the studied population's peripheral blood from which T lymphocytes were isolated and incubated with monoclonal antibodies. To evaluate antigens’ expression, a FACS Galibur flow cytometre was used; the evaluated cells were labeled with fluorochrome-conjugated monoclonal antibodies. Next, mononuclear cells were rinsed with cold PBS and suspended in the lysis buffer. In the obtained cell lysate the c cytochrome concentration was determined with the use of an immunoenzymatic test, HumanCytochrome cELISA Kit (BenderMed Systems, Austria). Obtained results were compared with the measurements taken in 20 healthy individuals who constituted the control group. On the basis of conducted study it was found that the level of c cytochrome concentration was significantly increased in the T CD3+ lymphocytes in the peripheral blood of patients with stage IV of laryngeal cancer. A positive correlatio was also found between the cytochrome level in lymphocytes and the advancing stage of the disease. Our own observation give grounds to conjecture that together with the progress of laryngeal cancer the energetic potential of lymphocytes increases and so does the readiness of the lymphatic cells to undergo the redox processes, therefore the amount of c cytochrome in the cells increases.     

An immunohistologic study of the distribution and status of activation of head and neck tumor infiltrating leukocytes

We examined tumor infiltrating leukocytes (TIL) in frozen sections of 28 biopsies from squamous cell carcinomas of the head and neck (SCCHN). In so doing, we used monoclonal antibodies (MoAb) directed against various leukocyte antigens. As defined by HLe-1+ cells, leukocyte infiltration was present in all biopsies. The amount of HLe-1+ cells was more often greater in stage III than in stage IV lesions. Most of the TIL were identified as CD5+ T-lymphocytes. In contrast, CD19+ B-cells were sparse in most biopsies. CD14+ monocytes/macrophages were found in only a few specimens. The relative proportion of CD4+ T-helper cells was higher than or at least equal to CD8+ suppressor/cytotoxic cells in all samples tested. Interleukin-2 (IL-2) receptor+ lymphocytes were evident in 13 of 22 biopsies stained for CD25 reactivity, and were more often observed in stage III than in stage IV tumors. All biopsies from recurrent tumors had no detectable IL-2 receptor+ cells. Our findings provide evidence for a positive correlation between a greater amount of TIL in earlier stages of SCCHN. The presence of IL-2+ lymphocytes suggests that SCCHN may be capable of activating resting lymphocytes for further IL-2-induced proliferation.     

Immunohistology of tumor tissue in local administration of recombinant interleukin-2 in head and neck cancer

Biopsied specimens from patients treated by local administration of rIL-2 were examined immunohistologically. Lymphocytes infiltrating into the cancer tissue before and after administration of rIL-2 were observed on paraffin embedded sections by Hematoxilin Eosin (HE) stain in relation to the clinical effect. And their subsets were identified on frozen sections by Avidin Biotin peroxidase Complex (ABC) method using monoclonal antibodies to examine what kinds of effector cells were induced and increased in the tumor site by rIL-2 in vivo. The intensity of increasing of tumor infiltrating lymphocytes (TIL) was correlated with the clinical effect of local administration of rIL-2. Tumor Infiltrating Lymphocytes were mainly T lymphocytes, of which Leu-2a+ cells (suppressor/cytotoxic T cells) and Leu-3a + 3b+ cells (helper/inducer T cells) were almost equally observed. Leu-2a+ cells were considered to be mainly cytotoxic T lymphocytes because few Leu-2a+ cells were stained with anti-Leu-15 antibody. After administration of rIL-2, marked infiltration of T lymphocytes was observed and there was no obvious different response in degree of infiltration between Leu-2a+ cells and Leu 3a + 3b+ cells. Furthermore, IL-2 receptor+ T lymphocytes (activated T lymphocytes) were increased after administration of rIL-2. And natural killer (NK) cells were slightly observed and also increased after administration of rIL-2. These findings suggest local administration of rIL-2 has possibility to induce cytotoxic T lymphocytes, to increase NK cells and to activate these lymphocytes in vivo.     

T and B lymphocyte rosettes in tonsils of atopic and non-atopic children.

This investigation was carried out to determine the immunological function of the human tonsils, especially the role of lymphocytes of atopic and non-atopic children. An E- and EAC-rosette forming technique is introduced, with semiquantitative evaluation of T and B lymphocytes in the peripheral blood of 56 normal subjects, 28 atopic and 5 non-atopic children of both sexes and different ages. The percentage of T and B lymphocytes in removed palatine tonsils in 28 atopic and 5 non-atopic children was calculated. The percentage of rosette-forming cells in E and EAC suspensions appeared to be independent of age and sex. The percentage of T lymphocytes in peripheral blood was significantly higher than in the palatine tonsils, whereas the percentage of B lymphocytes was significantly higher in the tonsils of atopic and non-atopic children than in the peripheral blood. The mean percentage of B lymphocytes in the tonsils of atopic children was significantly higher than in non-atopic children. The percentage of B lymphocytes was found to be significantly higher in hypertrophic than in small tonsils. Also, in the tonsils with adhesions there was a statistically significantly higher percentage of EAC rosettes than in the tonsils free of adhesions. The percentage of T lymphocytes was statistically significantly higher in the small tonsils.     

Prevalence of Epstein-Barr virus in tonsils and adenoids of United Arab Emirates nationals

Objective

Given that Epstein-Barr virus (EBV) often inhabits human tonsils and adenoids, it remains to be distinctively determined its prevalence and in which cell and microenvironment the virus is present.

Methods

To determine the prevalence of EBV in the tonsils and adenoids of the United Arab Emirates (UAE) nationals and to provide a basis for understanding the origin and biology of EBV-infected cells, the immunophenotype of all EBV-infected cells in 46 tonsils and 46 adenoids was determined by EBER in situ hybridization and immunohistochemistry with monoclonal antibodies to T cells (CD3), B cells (CD20), and epithelial cells (cytokeratin AE1/AE3), as well as immunostaining with antibodies to EBV latent membrane protein-1 (LMP-1).

Results

EBV was found in 43% of tonsillectomy specimens and 15% of adenoidectomy specimens. All EBV-infected cells were found to be B lymphocytes. About 90% of the infected B cells are found in the interfollicular regions of tonsils and adenoids and the remaining 10% are found within the follicles. There is no significant association between EBV infection, age (P = 0.324) and gender (P = 0.442).

Conclusion

EBV is associated with tonsillar hypertrophy and is prevalent in 43% of our cases. EBV is only detected in B lymphocytes and we believe that B lymphocytes are sites of primary infection and latency. In situ hybridization is the gold standard for the detection of EBV in tissue.     

Is immune system influenced by adenotonsillectomy in children?

OBJECTIVE: Tonsils and adenoids are lymphoid tissues that are located in the pharynx and play an important role against invading antigens of the upper respiratory tract. The present study analyses serum immunoglobulin levels and peripheral blood (PB) lymphocyte subsets in children, 24-48 h prior to and 4-6 weeks after adenotonsillectomy, in order to determine early effects of adenotonsillectomy on the immune system. METHODS: The study population consists of 15 children (aged 4-10 years) who underwent adenotonsillectomy because of adenoidal hypertrophy and chronic tonsillitis and 15 age-matched healthy children without a history of adenotonsillectomy. Serum IgG, IgA and IgM levels were measured by nephelometry. PB lymphocyte subsets were analysed by using monoclonal antibodies and flow cytometry. RESULTS: Children with chronic tonsillitis have increased levels of CD19+ B lymphocytes compared to healthy controls in the pre-operative period. The percentage of B lymphocytes bearing CD23 was found to be significantly higher in patients, most likely representing in vivo B lymphocyte activation due to chronic antigenic stimulation. After the adenotonsillectomy, despite ongoing B lymphocyte activation, CD8+ T lymphocyte levels increased and B cell levels returned to normal. A slight decrease in serum IgG, IgA and IgM levels was detected in the post-operative period compared to prior levels. CONCLUSION: Adenotonsillectomy performed in children leads to alterations that may reflect a compensatory response of the developing immune system after the removal of the lymphoid tissue in the setting of chronic antigenic stimulation. However, these changes do not cause significant immune deficiency.     

Lymphocyte subsets in normal airway mucosa of the human nose

The distribution and number of lymphocytes, monocytes/macrophages, and cells expressing HLA-DR antigen were studied in frozen biopsy sections of nasal mucosa from 40 healthy adults, using monoclonal antibody avidin-biotin immunoperoxidase techniques. The lymphocyte to monocyte/macrophage ratio was estimated to be 10:1; the T cell to B cell ratio was 3:1; and the T helper/inducer cell to T suppressor/cytotoxic cell ratio averaged 2.5:1. Regional differences were observed with a relatively increased number of T suppressor/cytotoxic cells around submucosal glands, and a relatively large number of B cells in lymphocyte aggregates in the lamina propria. The HLA-DR antigen was expressed in epithelial cells, suggesting involvement of surface epithelium of human airway in local immune responses.     

Type B haemophilus influenzae—induced otitis media in the mouse

To characterize the middle and inner ear cellular inflammatory responses to otitis media using im-munohistochemical methods, we inoculated type B Haemophilus influenzae into the middle ears of healthy adult BALB/c mice. Mac-1⁺ neutrophils and macrophages appeared in the middle ear at 3 days. Lyt-1⁺ T cells and Lyt-2⁺ T suppressor/cytotoxic cells entered the middle ear mucosa on days 7 and 14. IgG⁺ and IgM⁺ T cells were present at all time points, with IgA⁺ lymphocytes forming the majority of mucosal immunoglobulin-bearing cells at 2 weeks. The co-chlear scala tympani contained Lyt-1⁺ and Mac-1⁺ cells and two endolymphatic sacs stained diffusely with anti-IgA and -IgG antibodies. Lyt-1/L3T4⁺ T lymphocytes greatly outnumbered B lymphocytes, suggesting that helper/inducer T cells play a more important role in acute otitis media than has been recognized. Inner ear changes occurred after a single episode of otitis media.     

The T lymphocyte content of the efferent lymphatics of the human palatine tonsil

Human palatine tonsils have been studied by enzyme histochemical means for T lymphocyte marking enzymes (acid α-naphthyl acetate esterase and acid phosphatase). These enzymes show discrete dot like activity in T lymphocytes. An almost pure population of T cells (90–100% of cells present) is seen in the efferent lymphatics of all of the 12 tonsils studied. These findings are of probable immunological significance.     

Subsets of tonsillar lymphocytes and activated cells in each subset analysed by two-color flow cytometry

We analysed subsets of tonsillar lymphocytes and activated cells in each subset by two-color flow cytometry. There were many helper T cells (CD 4+ leu 8-) and few inducer T cells (CD 4+ leu 8+) in the tonsil. The situation was the reverse in the peripheral blood. The tonsil had few suppressor T cells (CD 8+ CD 11+). The proportion of activated cells (HLA-DR+) was low in the peripheral blood and high in the tonsil. In the tonsil, the ratio of cells with transferrin receptors to total lymphocytes was higher in the B than in the T subset, and higher in the subset of CD 4+ than in that of CD 8+ cells. Activated and proliferating lymphocytes were more abundant in the tonsils of children than in those of adults and more abundant in patients with hyperplastic tonsillitis than in those with recurrent tonsillitis.     

Lymphocyte subsets of maxillary mucosa in chronic inflammation

Subsets of infiltrating lymphocytes within maxillary sinus mucosae of patients with chronic sinusitis were investigated by immunoperoxidase staining of frozen sections with the use of monoclonal antibodies. The most commonly observed infiltrating cell type was suppressor/cytotoxic T cells (CD8+) and smaller subpopulations of lymphocytes were helper/inducer T cells (CD4+) and B cells (CD20+). Variable numbers of HLA-DR+ cells were commonly observed in the lamina propria. The fibrous type of chronic sinusitis was found to have more suppressor/cytotoxic T cells (CD8+) and lower CD4/CD8 ratio than the other histopathological types.     

Immunofluorescent assessment of tumour infiltrating cells in laryngeal carcinoma. Application of monoclonal antibodies

In order to gain some insight into host cell accumulations within primary tumour, frozen sections from surgical specimens of laryngeal carcinoma were subjected to indirect immunofluorescence using a panel of monoclonal antibodies against various human lymphocyte subsets as well as macrophages. In addition, polyclonal antibodies against Ig were used in order to trace B cells. Numerous host cell infiltrates seen at the tumour periphery were composed of T4 (helper) lymphocytes and macrophages. Lymphocytes of OKT8 (suppressor/cytotoxic) and Leu-7 (NK cells) series were intermingled with tumour cells in the case of scanty infiltrates. Infiltrating cells were also linked to the presence of metastases in regional lymph nodes. OKT4-positive abundant infiltrates were usually accompanied by uninvolved nodes, while scanty ones with OKT8 specificity were relatively frequently seen in the patients with evidence of nodal metastases. These differences were not statistically significant, however, B cells as well as plasma cells were infrequently observed and were encountered both in tumour samples with intensive cellular infiltrates as well as in those with scanty ones.     

Increased percentage of T cells with the expression of CD127 and CD132 in hypertrophic adenoid in children with otitis media with effusion

The hypertrophic adenoid may promote chronic suppurative otitis media in children as it fulfills its immune function. The number of lymphocytes in the adenoid and their cooperation in the immune response depend of on their proliferation and migration to the effector sites. Interleukin 7 (IL-7) is essential for the normal development and function lymphocytes. IL-7 plays pivotal role for activation and proliferation of T and B cells. The heterodimeric interleukin-7 receptor (IL-7R) is composed of the IL-7Rα (127) and the common cytokine receptor γc (CD132). The aim of this study was to evaluate the percentage of lymphocytes T (CD4⁺ and CD8⁺) with IL-7R (CD127 and CD132) expression in hypertrophic adenoid in children suffering with otitis media with effusion for a?duration of 3 months. Adenoid excised due to hypertrophy with or without chronic otitis media with effusion was used as study material. CD4⁺ CD127⁺, CD4⁺132⁺, CD8⁺CD127⁺ and CD8⁺CD132⁺ cell subpopulations were identified using monoclonal antibodies and flow cytometry. The percentage of CD4⁺ and CD8⁺ T cells with CD127 receptor expression in hypertrophic adenoid of children with otitis media with effusion was statistically significantly higher than in hypertrophic adenoid group. The percentage of CD4⁺ T cells with CD132 expression in the study group was statistically significantly higher than in the reference group. The percentage of CD8⁺ T cells with CD132⁺ expression was not statistically different in both groups. The increased percentage of T lymphocytes with IL-7R expression (CD127 and CD132) in hypertrophic adenoid seems to influence the quantity of lymphocytes and upset the immunological function of tonsils which can influence the course of otitis media with effusion.     

Clinical immunological study of T lymphocytes and EA rosette forming cells in tonsils.

Tonsillar E and EA rosette forming lymphocyte subpopulations were studied in 120 tonsillectomized patients. T cell ratios was usually lower in the tonsils than in the blood. EA binding cells were studied with indicator systems of human or rabbit antibody sensitized red cells, respectively. Poorly sensitized human RBCs (EArabbit) bind much better to tonsillar cells than to blood lymphocytes and so this system proved to be specially suitable to study tonsillar EA binding cells. Increase in E and EA rabbit rosette forming cell frequencies were found with the age of patients. Decrease in percentage of T cells and EA rabbit rosette binding cells were found with high frequency of acut tonsillitis and with clinical sysmptoms of chronic local inflammation. The variation of these lymphocyte subpopulations with the local tonsillar inflammatory processes suggest a considerable clinical immunological role of local T cells and of this portion of Fc receptor postive lymphocytes.