Fluoride resistant alpha-naphthyl acetate esterase and combined enzyme histochemistry in the study of normal and pathologic lymphoid tissues

Alpha-Naphthyl acetate esterase (ANAE) and fluoride resistant alpha-naphthyl acetate esterase (FRANAE) have been compared as histochemical methods to identify T lymphocytes in sections of normal and pathological human lymphoid tissues. In addition, the FRANAE method was combined with alkaline phosphatase (ALP) in order to simultaneously evaluate the relationship between T lymphocytes and fibroblastic reticular cells (ALP) positive). The "dot like" esterase positivity of T lymphocyte was better evaluated by using FRANAE when compared to ANAE because of fluoride inhibitor of the strong esterase activity of dendritic cells and most macrophages. The combined ALP-FRANAE method clearly demonstrated a large number of fibroblastic reticular cells within the T-areas in various normal and pathological tissues such as hyperplastic lymph nodes and especially in the lymph nodes and spleens from patients with Hodgkin's disease.     

Histochemical identification of human T lymphocytes from paraffin sections

The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

Summary The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25±0.27 mU/10⁷ cells and 6.18±0.87 mU/10⁷ cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10⁷ cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10⁷ cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10⁷ cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10⁷ cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

A novel recombinant protein of IP10-EGFRvIIIscFv and CD8+ cytotoxic T lymphocytes synergistically inhibits the growth of implanted glioma in mice

The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed on glioma cells and has been thought to be an excellent target molecule for immunotherapy. IP-10 is a potent chemokine and can recruit CXCR3⁺ T cells, including CD8⁺ T cells that are important for the control of tumor growth. This study is aimed at investigating the therapeutic efficacy of a novel fusion protein of IP10-EGFRvIIIscFv (IP10-scFv) in combination with glioma lysate-pulsed DCs-activated CD8⁺ cytotoxic T lymphocytes (CTLs) in a mouse model of glioma. A plasmid of pET-IP10-scFv was generated by linking mouse IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv, a (Gly₄Ser)₃ flexible linker and a His-tag. The recombinant IP10-scFv in E. coli was purified by affinity chromatography and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. C57BL/6 mice were inoculated with mouse glioma GL261 cells in the brain and treated intracranially with IP10-scFv and/or intravenously with CTL for evaluating the therapeutic effect. The glioma-specific immune responses were examined. The IP10-scFv retained anti-EGFRvIII immunoreactivity and IP-10-like chemotactic activity. Treatment with both IP10-scFv and CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, accompanied by increasing the numbers of brain-infiltrating lymphocytes (BILs) and the frequency of CXCR3⁺CD8⁺ T cells, enhancing glioma-specific IFN-γ responses and cytotoxicity, and promoting glioma cell apoptosis in mice. Our novel data indicate that IP10-scFv and CTL have synergistic therapeutic effects on inhibiting the growth of mouse glioma in vivo.     

Antibody-dependent cellular cytotoxicity in the rat, rabbit, and guinea pig: relationship of killer (K) cell activity with the presence of Fc+ C3- and Fc+ C3+ lymphocytes.

ADCC of lymphocyte suspensions from the spleen, lymph node, and blood in the rat, rabbit, and guinea pig, was directly correlated with the presence of Fc⁺ C₃^? and indirectly with the presence of Fc⁺ C₃⁺ lymphocyte subpopulations in these preparations. Rabbit lymphocytes, from all three lymphoid compartments, were deficient in the Fc⁺ C₃^? subpopulation but enriched in the Fc⁺ C₃⁺ subpopulation and lacked K-cell activity. A similar lymphocyte profile and lack of K-cell activity was found in the lymph node of the rat and guinea pig. Lymphocytes from the blood and spleen, on the other hand, had a substantial population of Fc⁺ C₃^? lymphocytes and a prominent K-cell activity. The Fc⁺ C₃⁺ lymphocyte, although unable to lyse IgG-coated target cells, is able to compete in vitro with the Fc⁺ C₃^? (K) cell and significantly reduce ADCC. Phagocytic cells lyse very effectively erythroid target cells and contamination by such cells may explain previously reported K-cell activity of rabbit lymphocytes.     

Histological and enzyme histochemical investigation of the hemal nodes of the hair goat

Abstract

We investigated the structure of the hemal node in six healthy hair goats using histological and enzyme histochemical methods. After processing, tissue sections were stained with Crossman's trichrome, Gordon-Sweet's silver and Pappenheim's panoptic stains. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were demonstrated in frozen sections. Hemal nodes were encapsulated by connective tissue and few smooth muscle cells. Several trabeculae originated from the capsule and extended into the hemal node. A subcapsular sinus was present beneath the capsule and was continuous with the deeper sinuses. Subcapsular and deep sinuses were filled with erythrocytes. The parenchyma consisted of lymphoid follicles, diffuse interfollicular lymphocytes and irregular wide lymphoid cords. Cortical and medullary regions were not distinct. ANAE (+) and ACP-ase (+) cells were located mainly in the germinal centers of the lymphoid follicles and also were scattered equally in the interfollicular region and lymphoid cords. Monocytes, macrophages and reticular cells displayed a diffuse positive reaction, whereas localized granular positivity was observed in lymphocytes. We demonstrated that the general structure of the hair goat hemal nodes is similar to that of other ruminant species.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Sister-chromatid exchange in highly purified human CD4 and CD8 lymphocytes

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

Histochemical study on human germinal centre, mantle-zone and extra-follicular area lymphoid cell subpopulations. Immunological and cytochemical correlations with lymphomatous cells, peripheral normal and leukemic lymphocytes

Tissue sections of lymph nodes, appendices and tonsils, together with smears of immunologically separated peripheral lymphoid cells from a B-CLL and lymphomatous cells from an immunocytoma were submitted to combined enzyme cytochemical investigations with acid alpha-naphthyl acetate esterase (ANAE), beta-glucuronidase (B-G), acid phosphatase (AcPh), adenosine triphosphatase (ATPase), a,d 5'nucleotidase (5'N). T-cells were Acph+, ATPase- and 5'N-. The vast majority of T- and B-cells displayed ANAE and B-G activities with two distinct staining patterns (T-like and B-like pattern). A high proportion of lymphoid cells in the germinal centre (G.C.) and the vast majority of lymphoid cells in the mantle-zone (M.Z.) were shown to belong to B-cell system because of the expression of ATPase and 5'N in their membranes. Some lymphoid cells positive for ANAE and B-G with a B-like pattern and for AcPh were recognizable in the G.C. In the M.Z. only a few lymphoid cells being ANAE+, with a T-like pattern, and AcPh+ were shown to belong to the T-cell system. In contrast, in this zone a high proportion of small lymphoid cells (64% +/- 10%) showed ANAE activity, mostly with B-like pattern. Therefore, these findings indicate that in the M.Z. a high proportion of B-cells ATPase+ and 5'N+ also display ANAE activity. By comparison of the results obtained from lymphoid tissue sections, B-CLL and immunocytoma cell suspensions and normal circulating lymphocytes we can conclude that B-ANAE-positive cells of the M.Z. do not usually appear in the peripheral blood. They circulate in large numbers only in some pathological conditions (like our reported B-CLL). Therefore, B-ANAE-positive lymphoid cells of the mantle, with a B-like staining pattern, include a wide range of subsets which exclude large lymphoid cells and plasma cells.     

Modulation of the adherence of human lymphocytes to measles-infected cells by prostaglandin E1: A differential effect on lymphocyte subpopulations

Prostaglandins of the E series (PGE) may serve as important regulators of human immune responsiveness. The present study was designed to examine the possibility that PGE may effect human lymphocyte function by the modulation of surface receptors. The presence of surface binding sites on human lymphocytes for measles virus antigens was studied using a rosette adherence assay. We observed that the addition of PGE₁ increased the proportion of measles-infected cells (Hela-Kll) with adherent lymphocytes (75% increase at 3 × 10⁻⁶ M PGE₁). PGE was observed to enhance the adherence of purified normal peripheral T cells (87%) and T lymphoid cells (Molt 3) (27%). In contrast, no significant change in normal peripheral B cell or B lymphoid cell (Raji) adherence was observed with the addition of PGE. These results are consistent with a selective modulation of surface measles virus binding sites by PGE₁ on T and not B lymphoid cells.     

Enzymatic relationship between human Tγ+ lymphocytes and thymocytes

The lactate dehydrogenase isoenzyme pattern has been determined in human thymocytes and in peripheral blood T lymphocytes, Tγ⁺, and Tμ⁺ lymphocytes. Thymocytes have a highly significant lower activity in LDH-1 and LDH-2 together with a highly significant higher activity in LDH-3 and LDH-4 than peripheral T lymphocytes. The same differences are seen when Tγ⁺ and Tμ⁺ cells are compared. On the contrary there are almost no differences in pattern between the peripheral T lymphocytes and Tμ⁺ cells on one hand and between thymocytes and Tγ⁺ cells on the other hand. These findings suggest that both thymocytes and Tγ⁺ cells exhibit a very immature LDH pattern with regard to the Tμ⁺ cell population.     

Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. I. Separation into subsets of lymphocytes bearing distinctive markers

Lymphocytes from human peripheral blood have been separated by countercurrent distribution in a charged aqueous two-phase system composed of Dextran T 500 and polyethylene glycol 6000 with a cell yield of 59–88% and viability above 90%. A highly reproducible partition pattern was seen with four distinct peaks. Lymphocytes with surface membrane immunoglobulin (SmIg) were located in the first part of the distribution corresponding mainly to peak I. T lymphocytes as detected by E rosetting and α-naphthyl acetate esterase (ANAE) staining showed a broad distribution with a maximum in peaks II and III. ANAE-negative lymphocytes were seen in both extremes of the distribution, corresponding to B cells in the first part and to a population of E^? and SmIg^? lymphocytes in the last part. Monocytes were present in all fractions with some enrichment in peaks II–IV. Lymphocytes with low-affinity Fc receptors were found in B-cell-containing fractions in the first part of the distribution, but also in the last part. Lymphocytes with high-affinity Fc receptors were detected mainly in peak IV. It is thus demonstrated that peripheral blood lymphocytes can be fractionated into subpopulations enriched in cells with characteristic markers.     

CXCL12 is a costimulator for CD4+ T cell activation and proliferation in chronic lymphocytic leukemia patients

Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4⁺ T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4⁺ T cells from CLL patients, similarly in T cells from ZAP-70⁺ to ZAP-70^? patients. Autologous nurse-like cells establish a close contact with CD4⁺ T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.     

PNA-binding glycans are expressed at high levels on horse mature and immature T lymphocytes and a subpopulation of B lymphocytes

In mammals, the binding of peanut agglutinin (PNA) on the plasma membrane defines subpopulations among lymphocytes from peripheral blood and lymphoid organs. PNA binds Gal 1,3GalNAc residues provided that they are not sialylated. Here, we studied the expression of PNA-binding glycans on healthy horse peripheral blood, thymus, lymph node and spleen lymphocytes. We first demonstrated the binding specificity of PNA for galactose residues by competition experiments and the inhibitory role of sialic acids in PNA binding by sialidase digestion. Unlike human and murine lymphocytes, all equine lymphocytes were found positive by flow cytometry analysis. Double-staining analyses showed that lymphocytes expressing high levels of PNA-binding glycans (PNA^high lymphocytes) were made up of the great majority of CD5⁺, CD4⁺ and CD8⁺ cells, and of 30 and 50% of sIg-bearing lymphocytes in peripheral blood and in lymph nodes or spleen, respectively. Lectin histochemistry suggested that lymph node germinal centres contained PNA^high B cells. Contrary to what is found in humans and mice, PNA staining intensity on CD5⁺, CD4⁺ and CD8⁺ cells did not differentiate immature from mature T lymphocytes in the equine thymus. The functional consequences of these differences are discussed. Published in 2005.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections

Summary An improved histochemical technique for the demonstration of lactate dehydrogenase activities in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of lactate dehydrogenase into the medium during incubation. In the histochemical system the NAD⁺-dependent enzyme catalyzes the electron transfer from lactate into NAD⁺. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Amytal is incorporated into the incubating-medium to block electron transfer to the cytochromes. Problems involved in the histochemical demonstration of lactate dehydrogenase activity are discussed.This investigation was in part supported by a grant from the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody

Development of T lymphocytes in Xenopus laevis was studied using a mouse monoclonal antibody (mAb), XT-1, that was produced against surface determinants on thymocytes of J strain frog. Ontogenic studies, employing immunofluorescence, showed that cells positive for the determinant recognized by XT-1 mAb (XT-1⁺ cells) were first detected in the thymus of J strain Xenopus by Nieuwkoop and Faber stage 48 (7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days postfertilization). Percentages of XT-1⁺ cells in the thymus increased rapidly by stage 49 (10 days postfertilization) and reached adult levels by stage 52, and those in the spleen, liver, and kidney reached adult levels by stage 56 (40 days postfertilization). Electron microscopic immunohistochemistry revealed that most XT-1⁺ cells in thymuses from stage 56 larvae were typical small lymphocytes (4–7 μm in diameter). In contrast, many XT-1⁺ cells in larval thymuses at stage 49 are large (8–10 μm in diameter) lymphoblastoid cells. Thymectomy at stage 46 (5 days postfertilization) depleted XT-1⁺ cells in larval and adult lymphoid organs to background levels. These results suggest that the XT-1⁺ cells are differentiated from the lymphoid precursor cells in the thymus before the appearance of small lymphocytes and migrate into peripheral lymphoid organs. The cell surface determinant recognized by the XT-1 mAb may provide an important marker for the differentiation of T lymphocytes in Xenopus.     

HIV-specific cytotoxic T lymphocytes against alveolar macrophages: specificities and downregulation

To analyse the evolution of alveolar-lymphocyte-mediated cytotoxic activity directed against autologous alveolar macrophages (AM), cytotoxic assays against various HIV⁺ target cells were performed in a cohort of 75 patients with HIV-associated lymphoid interstitial pneumonitis (LIP) studied at distinct stages of HIV infection. Our data confirm that alveolar HIV-specific cytotoxic T lymphocytes (CTL) against AM were detectable before AIDS in patients with CD8⁺ LIP. Mild CD8⁺ lymphocytic alveolitis occurs silently in 62% of stage II and III patients with no respiratory symptoms. In these cases, the lack of spontaneous alveolar-lymphocyte-mediated cytotoxic activity against autologous AM may contrast with the detection of primary alveolar CTL specific for HIV proteins such as nef. In AIDS patients, the alveolar CTL lytic efficiency against both AM- and HIV-antigen-expressing cells can be inhibited by a suppressore factor produced by alveolar CD8⁺ CD57⁺ cells. Therefore, spontaneous CTL lysis of AM may be (1) limited to a subgroup of patients with active LIP and (2) controlled by distinct mechanisms, including suppressor phenomenons, and HIV replication levels in AM.     

Macrophage and dendritic cell infiltration in head and neck squamous-cell carcinoma; an immunohistochemical study

A study was undertaken to help us reach a better understanding of the tumor-infiltrating pattern of lymphoid cells and in particular of monocyte-derived cells, namely the CD68⁺, acid-phosphatase-expressing scavenger macrophages and the MHC-class-II- and S100-antigen-presenting dendritic cells in head and neck squamous-cell carcinoma. In the stroma of the tumors distinctive small fields of lymphocytes were found, the T cell areas of these fields being intermingled with dendritic cells. Intra-epithelial dendritic cell infiltration was low. The infiltrative pattern of macrophages was similar to patterns described in earlier studies with substantial stromal invasion and inconsistent intra-epithelial invasion, but small granuloma-like structures of CD68⁺ macrophage-like cells, found in the stroma of tumors, have not been reported before. The histochemical localization of the tumor-infiltrated dendritic cells and macrophages supports the view that the former cells are involved in the sensitization to tumor antigens, whereas the latter cells are involved in tumor cytotoxicity/scavenging of tumor cell debris. Although it has been shown in the past that transmembranal (TM) factors (p15E-like factors) present in the serum and tumor of patients with cancer of the head and neck have suppressive effects on monocyte/macrophage/dendritic cell function, a relationship between the intensity of epithelial staining for TM factors and the infiltrative pattern of monocytes/macrophages/dendritic cells could not be demonstrated.     

High Expression of Tumor HLA-DR Predicts Better Prognosis and Response to Anti-PD-1 Therapy in Laryngeal Squamous Cell Carcinoma

BackgroundHLA-DR is expressed in epithelial and several types of tumor cells. However, the correlation between tumor-expressed HLA-DR (teHLA-DR) and patient outcome as well as its regulation on the tumor microenvironment (TME) of laryngeal squamous cell carcinoma (LSCC) are yet to be elucidated.MethodsHematoxylin and eosin (HE) staining were performed to define the tumor nest and stroma of LSCC tissue microarrays. teHLA-DR tumor cell, CD4⁺ and CD8⁺ tumor-infiltrating T lymphocytes (TITLs) were obtained and analyzed through double-labeling immunofluorescence and immunohistochemical staining. The recurrence-free (RFS) and overall survival (OS) curves were plotted using the Kaplan-Meier method and tested by the log-rank test method. Expression of teHLA-DR⁺ tumor cells and infiltration of T lymphocytes and their corresponding subgroups were analyzed by flow cytometry using fresh LSCC tissue samples.ResultsOur research discovered elevated expressions of multiple MHC-II-related genes in tumor compared to the adjacent normal tissue samples of LSCC patients. We also found that patients in the teHLA-DR high-expression group (teHLA-DR^high) tend to have less tumor recurrence and better survival outcomes compared to those in the teHLA-DR^low group. Intriguingly, teHLA-DR⁺ tumor cells had significantly higher PD-L1 and PD-L2 expression and their TME showed increased infiltrated T lymphocytes (TITLs). Flow cytometry analysis and IHC staining indicated that CD4⁺ TITLs but not CD3⁺ total TITLs or CD8⁺ TITLs were significantly enriched in teHLA-DR⁺ tumors.ConclusionsteHLA-DR may be a predictive marker for favorable prognosis and response to anti-PD-1/PD-L1 therapy of LSCC, possibly due to the increased CD4⁺ TITLs in the TME.     

Murine spleen lymphocytes bearing receptors for peanut agglutinin: VII. Separation and functional characterization

Murine spleen lymphocytes having receptors for peanut agglutinin (PNA) were visualized by a specific rosetting technique. The number of lymphocytes forming PNA rosettes (PNA_R⁺) is dependent on the PNA concentration used. T lymphocytes are the primary PNA-rosetting lymphocytes for low PNA concentration (2.5 μg/ml), while both T and B lymphocytes are involved in high PNA concentration (10 μg/ml). Functional studies show that when spleen lymphocytes are separated at a low PNA concentration, the PNA_R⁺ do not respond to T mitogens and suppress antigen-specific immune responses. However, when spleen cells are separated at a high PNA concentration, the PNA_R⁺ do not exhibit differential functional properties as compared to non-PNA-rosetting lymphocytes. The implication of these findings is discussed.