Age-related alterations of the CD19 complex and memory B cells in children with Down syndrome

Children with Down syndrome (DS) have a high incidence of recurrent respiratory tract infections, leukaemia and autoimmune disorders, suggesting immune dysfunction. The present study evaluated the role of the CD19 complex and memory B cells in the pathogenesis of immunodeficiency in children with DS. The expression levels (median fluorescein intensity—MFI) of CD19, CD21 and CD81 molecules on the surface of B cells and memory B cell subsets were studied in 37 patients and 39 healthy controls. Twenty-nine of the DS group had congenital cardiac disease. The B cell count was significantly low in children with DS compared with healthy age-matched controls for all three age groups (under 2 years; 2–6 years and older than 6 years). The MFI of CD19 was reduced in all the age groups, whereas that of CD21 was increased in those older than 2 years with DS. The expression level of CD81 was significantly increased in those older than 6 years. Age-related changes were also detected in memory B cell subsets. The frequency of CD27⁺IgD⁺IgM⁺ natural effector B cells was reduced in children with DS who had needed hospitalisation admission due to infections. The observed intrinsic defects in B cells may be responsible for the increased susceptibility of children with DS to severe respiratory tract infections.     

Human IgM+IgD+ B cells,the major B cell subset in the peripheral blood,express Vϰ genes with no or little somatic mutation throughout life

Peripheral blood B cells of a 67-year-old person were separated into IgM⁺IgD⁺, IgM⁺IgD^?, and IgM^?IgD^? subsets, and nucleotide sequences of expressed immunoglobulin light chain variable (V) regions encoded by V?3 and V?4 gene family members were determined from amplified cDNA. V region sequences from IgM⁺IgD⁺ cells (the major B cell population in the blood) showed no or little somatic mutation (0.3%), in contrast to V? sequences from IgM⁺IgD^? and IgM^?IgD^? B cells (2.0% and 3.9%, respectively). This suggests that in the human like in the mouse, and independently of age, somatically mutated memory B cells accumulate in the compartment of IgM^?IgD^? cells, whereas the IgM⁺IgD⁺ subpopulation consists of cells whose antibody repertoire is mainly determined by V region gene rearrangements and N-region insertion, at the molecular level. The somatically mutated IgM+IgD^? cells may represent early descendants of IgM⁺IgD⁺ cells recruited into the memory cell compartment.     

Particularity of Local Immunity in the Nasopharynx

Immunological functions of the pharyngeal tonsil, palatine tonsils and blood leucocytes of children undergoing tonsillectomy were evaluated by determining T or B lymphocytes, the response to mitogens, and the cell-mediated immunological responses to tuberculin. In all the test systems used similar results were obtained with cells derived from either the palatine or pharyngeal tonsils. The mean percentage of T lymphocytes was significantly higher in the peripheral blood than in tonsils, but the reverse was true of B lymphocytes. The reaction to PHA was lower in tonsillar cell culture than in blood cell culture, but tonsillar cells reacted better to Con A than blood cells. In lymphocyte transformation tests tonsillar cells reacted to specific antigen (tuberculin) and this reaction was significantly higher than that of the parallelly tested blood lymphocytes. Further, in about 50% of the children tested, tuberculin caused migration inhibition of the mixture containing tonsillar cells and guinea pig peritoneal cells. Surprisingly, nearly identical results were obtained if migration inhibition test was performed with tonsillar cells alone. Consequently, poorly migrating tonsillar cells are nevertheless usable for direct migration inhibition testing.     

More newly formed T than B lymphocytes leave the intestinal mucosa via lymphatics

Many lymphocytes are produced in the intestinal mucosa, especially in the Peyer's patches. These newly formed lymphoid cells leave the gut wall, undergo further maturation and many reach the lamina propria of the intestinal mucosa where they function as effector and regulator cells of the intestinal immune response. However, the number and subset composition of these wall are not known. Therefore, the intestinal lymph duct was cannulated in eight minipigs, in which the mesenteric lymph nodes had been removed 3 months earlier. Thus, it was possible to obtain all lymphocytes leaving the intestinal mucosa including the Peyer's patches via lymphatics. The hourly output of lymphocyte subsets was examined over the course of 93 h. The percentage and the absolute numbers of newly formed T cells (CD2⁺, CD8⁺) and B cells (IgA⁺, IgM⁺) were determined by examining the incorporation of the DNA precursor bromodeoxyuridine. After a single i.v. bromodeoxyuridine injection 8.5% of the T, 55% of the IgA⁺ and 25% of the IgM⁺ cells were labeled. In absolute numbers (1.9 ± 0.7) × 10⁶ newly formed T cells, (0.4 ± 0.3) × 10⁶ IgA⁺ cells and (0.5 ± 0.4) × 10⁶ IgM⁺ cells emigrated from the gut wall per hour. Both T and B lymphocyte subpopulations that are produced in the intestinal mucosa leave the gut wall via lymphatics; interestingly, the T cells outnumber the B cells. Obviously the induction and maintenance of mucosal immunity depend to a large extent on the function of newly formed T lymphocytes emigrating from the Peyer's patches and/or from the mucosa without Peyer's patches.     

The surface epithelium of recurrent infected palatine tonsils is rich in γδ T cells

Using a large panel of MoAbs in quantitative morphometric analysis of immunohistochemically stained tissue sections, we compared the frequency and distribution of immune cells in palatine tonsils from patients with recurrent tonsillitis (RT) and patients with idiopathic tonsillar hypertrophy (ITH). We found that differences between the two patient groups in leucocyte populations were limited to the surface epithelium, whereas the cellular composition of interfollicular and follicular areas was similar. Most intraepithelial lymphocytes were CD8⁺ T cells in both groups. However, the number of intraepithelial T cells was significantly higher in RT compared with ITH. This was due to a selective increase in the number of intraepithelial CD8⁺γδ T cells utilizing Vδ1 and Vγ9. In both patient groups the majority of the intraepithelial γδ T cells expressed Vδ1 and Vγ9. Subepithelially, γδ T cells utilizing Vγ9 dominated over cells utilizing Vγ8, while equal proportions expressed Vδ1 and Vδ2. These results suggest that cells utilizing the otherwise rare combination Vδ1/Vγ9 in their T cell receptors (TCR) may constitute a major γδ T cell population in palatine tonsils and are probably reactive to antigens specific to the tonsillar milieu. Furthermore, they indicate that preferentially this γδ T cell subpopulation is involved in immune reactions within the surface epithelium in RT. We speculate that γδ T cells are involved in clearing infectious bacteria at the tonsillar surface and in limiting inflammatory responses in the tonsils. Both local expansion and infiltration of blood cells probably contribute to the high numbers of γδ T cells in RT patients.     

Scarcity of autoreactive human blood IgA+ memory B cells

Class‐switched memory B cells are key components of the “reactive” humoral immunity, which ensures a fast and massive secretion of high‐affinity antigen‐specific antibodies upon antigenic challenge. In humans, IgA class‐switched (IgA⁺) memory B cells and IgA antibodies are abundant in the blood. Although circulating IgA⁺ memory B cells and their corresponding secreted immunoglobulins likely possess major protective and/or regulatory immune roles, little is known about their specificity and function. Here, we show that IgA⁺ and IgG⁺ memory B‐cell antibodies cloned from the same healthy humans share common immunoglobulin gene features. IgA and IgG memory antibodies have comparable lack of reactivity to vaccines, common mucosa‐tropic viruses and commensal bacteria. However, the IgA⁺ memory B‐cell compartment contains fewer polyreactive clones and importantly, only rare self‐reactive clones compared to IgG⁺ memory B cells. Self‐reactivity of IgAs is acquired following B‐cell affinity maturation but not antibody class switching. Together, our data suggest the existence of different regulatory mechanisms for removing autoreactive clones from the IgG⁺ and IgA⁺ memory B‐cell repertoires, and/or different maturation pathways potentially reflecting the distinct nature and localization of the cognate antigens recognized by individual B‐cell populations.     

Acute wrist and foot drop associated with hepatitis C virus related mixed cryoglobulinemia: Rapid response to treatment with rituximab

The nature of the B-cell subsets associated with chronic hepatitis C virus related type II mixed cryoglobulinemia (HCV-MC) is unclear.We report the case of a 64-year-male with acute onset wrist drop and foot drop, secondary to HCV-MC related mononeuritis multiplex, who was treated with rituximab, an anti-CD20⁺ antibody directed against B cells. We monitored the frequency of B-cell subsets in peripheral blood before and after rituximab, and correlated B-cell subset changes with clinical response.Significant improvements in his wrist and foot drop, as well as his vasculitic rash, depression and erectile dysfunction were evident within six days of starting rituximab and have persisted several months after B-cell recovery.More than 95% of CD20⁺ B cells had disappeared from peripheral blood within 1 week, returning to baseline by week 21. CD20⁺CXCR3⁺ frequency at baseline was similar to that at week 21. CD20⁺CD5⁺, the human equivalent of B1 B cells and CD20⁺IgM⁺IgD⁺, naïve B cells were increased. By contrast, CD20⁺CD27⁺ memory cell frequency was reduced.These data suggest that CD27⁺ memory B cells, but not CD5⁺ and IgM⁺IgD⁺ B cells may play a role in the clinical manifestations of cryoglobulinemia.     

CD73 expression identifies a subset of IgM+ antigen‐experienced cells with memory attributes that is T cell and CD40 signalling dependent

B‐cell memory was long characterized as isotype‐switched, somatically mutated and germinal centre (GC)‐derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B‐cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype‐switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T‐dependent versus T‐independent responses. We report that CD73 expression identifies a subset of antigen‐experienced IgM⁺ cells that share attributes of functional B‐cell memory. This subset is reduced in the spleens of T‐cell‐deficient and CD40‐deficient mice and in mixed marrow chimeras made with mutant and wild‐type marrow, the proportion of CD73⁺ IgM memory is restored in the T‐cell‐deficient donor compartment but not in the CD40‐deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age‐associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B‐1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC‐associated memory generation during B‐cell differentiation, CD40‐signalling can influence the composition of the unswitched memory B‐cell pool. They also raise the possibility that a fraction of ABCs may represent T‐cell‐dependent IgM memory.     

Subepithelial B cells in the human palatine tonsil. I. Morphologic,cytochemical and phenotypic characterization

This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5–10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5⁻ B cells had a surface phenotype (IgM⁺, IgD⁺, CD23⁻, CD38^±, CD10⁻, CD44⁺) that was different from that of FM (IgM⁺, IgD⁺, CD23⁺, CD39⁺, CD38⁻, CD10⁻, CD44^±) and of germinal center (GC) (CD23⁻, CD39⁻, CD38⁺, CD10⁺, CD44^±, IgG⁺) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5⁻ B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5⁻ B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5⁻ B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5⁻ B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP⁺, while GC cells were consistently ALP⁻. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5⁻ B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5⁻ B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.     

B-lymphocyte subpopulations are equally susceptible to Epstein-Barr virus infection,irrespective of immunoglobulin isotype expression

While Epstein–Barr virus (EBV) is known to establish latency in the memory B‐cell compartment, there is controversy as to whether the memory or the naïve B cell is the initial target for infection. Here we have explored the infectability of the B‐cell subsets contained in peripheral blood and tonsils, as distinguished by their surface expression of the immunoglobulin isotypes that help to define naïve and memory pools. First we show that both CD21 and major histocompatibility complex (MHC) class II molecules – respectively, the major receptor and co‐receptor for EBV on B cells – are expressed at similar levels on blood and tonsillar B cells, irrespective of surface immunoglobulin class, indicating that each of the subsets demonstrate an equal potential, at least for infection. Then, following in vitro infection of total tonsillar B cells, we found that the relative frequencies of immunoglobulin (Ig)M‐, IgG‐ and IgA‐positive cells containing EBV‐encoded Epstein–Barr virus nuclear antigen 5 (EBNA5) protein at 48 hr were similar to those of the starting population. However, IgD expression was uniformly decreased, probably as a consequence of cellular activation. These data indicate that recirculating B cells have both the potential for, and susceptibility to, initial infection by EBV, irrespective of the immunoglobulin isotype expressed.     

A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation

Toll‐like receptors (TLRs) recognize specific pathogen‐associated molecular patterns (PAMPs), which subsequently trigger innate immunity. Recent data also suggest a role for TLRs in the direct activation of adaptive immune cells. In the present study, the expression and function of TLR1–TLR10 were characterized in purified human tonsillar B cells, focusing on differences among CD19⁺ CD38^– CD27^– (naïve B cells), CD19⁺ IgD^– CD27^–[germinal centre (GC) B cells] and CD19⁺ CD38^– CD27⁺ (memory B cells) cells. The study was also designed to compare the TLR expression in B cells obtained from infected and hyperplastic tonsils that served as controls. The results demonstrated a distinct repertoire of TLRs, in which TLR1, TLR2, TLR7, TLR9 and TLR10 predominated. No differences were found among naïve, GC and memory B cells. Tonsillar infection did not substantially alter the TLR expression profile in ex vivo‐isolated B‐cell subsets. Purified CD19⁺ B cells stimulated with Pam₃CSK₄, R‐837 and CpG oligodeoxynucleotide (ODN) 2006, via TLR1/TLR2, TLR7 and TLR9, respectively, showed an induction of interleukin‐6 secretion and an up‐regulated expression of human leucocyte antigen (HLA)‐DR. Collectively, the present study demonstrates that B cells exhibit constitutively high levels of specific TLRs, which are not developmentally regulated during the B‐cell differentiation process. Ongoing microbial infections, such as chronic tonsillitis, do not appear to affect the TLR profile in B cells. Furthermore, the distinct expression of TLRs allows B cells to respond directly to the cognate PAMPs. This further emphasizes the role of TLRs in directly activating adaptive immune cells.     

Expression of Three Immunoglobulin Isotypes by Individual B Cells During Development: Implications for Heavy Chain Switching*

ABSTRACT: Previous studies in mice and humans have shown that the first IgA⁺ and IgG⁺ B cells appearing during ontogeny also bear IgM on the surface. The acquisition of IgD seems to occur at a later stage in differentiation. In this study we have combined autoradiography with two-color immunofluorescence to directly detect human B cells expressing three surface isotypes. We report that the vast majority of IgA⁺ cells in the neonate also bear IgM and IgD; the same holds true for IgG⁺ cells. This phenotype is peculiar of newborns, while in the adult the majority of IgA⁺ and IgG⁺ cells are single. We discuss the genetic implications of such a finding for heavy chain switching.     

Identification of a novel subpopulation of germinal center B cells characterized by expression of IgD and CD70

CD27, which belongs to the tumor necrosis-factor receptor family, is expressed on germinal center (GC) but not on naive B cells, suggesting an important function of this molecule in the regulation of the GC reaction. We described here the expression of CD70, which is the ligand for CD27. We observed that in most tonsils, CD70 is only expressed on part of the IgD^?, CD38^? B cell population, which have been described as memory B cells. However, in 10 % of the tonsils tested, CD70⁺ IgD⁺ GC were found. The CD70⁺ GC B cells were small cells that also expressed CD44 and CD39, but were CD10^? and CD38^?, suggesting that they represent very recent immigrants that are in the process of forming a GC. The concordant expression of CD27 and its ligand CD70 on this primordial subset of GC B cells suggests an important role for CD27/CD70 interaction at this stage of GC formation.     

Intratonsillar detection of 27 distinct viruses: A cross-sectional study

Palatine tonsils have been observed to harbor several distinct respiratory and herpesviruses in separate studies. In this study, the presence of these viruses in palatine tonsils was comprehensively studied in both children and adults. A cross-sectional analysis of 181 patients (median age 22 years; range, 2.6-66) operated for a benign tonsillar disease was conducted. Real-time polymerase chain reaction was performed to detect 27 distinct viruses in all: eight human herpesviruses, 16 respiratory viruses, parvo B19, and polyoma BK/JC viruses. Clinical characteristics of the patients and underlying conditions were evaluated. In total, 92% of patients had virus detected in tonsils (Epstein-Barr virus 72%, human herpesvirus 7, and 6B 54% and 16%, respectively, enterovirus 18%, parvovirus B19 7% and the rest <4%). No herpes simplex virus 2, varicella zoster virus, polyoma JC virus, parainfluenza-, metapneumo-, or coronaviruses were found. Enterovirus was more common in children and was frequently observed in the presence of HHV6B. None of the viruses showed a positive association to the tonsillar disease. Respiratory symptoms were not associated with the prevalence of viruses. This study comprehensively reports a cross-sectional view of intratonsillar virus infections in elective tonsillectomy patients in a wide age range cohort. Tonsils are a major virus reservoir for distinct herpes and respiratory viruses without a positive association with tonsillar disease or respiratory symptoms.     

The association of sore throat and psoriasis might be explained by histologically distinctive tonsils and increased expression of skin‐homing molecules by tonsil T cells

Recent studies have highlighted the involvement of the palatine tonsils in the pathogenesis of psoriasis, particularly among patients with recurrent throat infections. However, the underlying immunological mechanism is not well understood. In this study we confirm that psoriasis tonsils are infected more frequently by β‐haemolytic Streptococci, in particular Group C Streptococcus, compared with recurrently infected tonsils from patients without skin disease. Moreover, we show that tonsils from psoriasis patients contained smaller lymphoid follicles that occupied a smaller tissue area, had a lower germinal centre to marginal zone area ratio and contained fewer tingible body macrophages per unit area compared with recurrently infected tonsils from individuals without skin disease. Psoriasis patients' tonsils had a higher frequency of skin‐homing [cutaneous lymphocyte‐associated antigen (CLA⁺)] CD4⁺ and CD8⁺ T cells, and this correlated significantly with their frequency of blood CLA⁺ T cells. The psoriasis patients also had a higher frequency of tonsil T cells expressing the interleukin (IL)‐23 receptor that was expressed preferentially by the CLA⁺ T cell population. In contrast, recurrently infected tonsils of individuals without skin disease had a higher frequency of tonsil T cells expressing the activation marker CD69 and a number of chemokine receptors with unknown relevance to psoriasis. These findings suggest that immune responses in the palatine tonsils of psoriasis patients are dysregulated. The elevated expression of CLA and IL‐23 receptor by tonsil T cells may promote the egression of effector T cells from tonsils to the epidermis, suggesting that there may be functional changes within the tonsils, which promote triggering or exacerbation of psoriasis.     

IgD class switching: Identification of a novel recombination site in neoplastic and normal B cells

IgD on normal B lymphocytes usually is co-expressed with IgM. A minority of normal plasma cells and rare B cell malignancies express exclusively IgD (IgM^?-IgD⁺). The low frequency has been explained by the lack of a recognizable switch region within the Cμ-Cδ intron. We analyzed four cases of IgM^?IgD⁺ hairy cell leukemia (HCL) by Southern (DNA) blot analysis and identified two cases with a recombinatorial event within the Cμ-Cδ intron and deletion of Cμ. DNA sequence analysis of junctional regions showed that Sμ or the immediate upstream region was used as a donor site and that the Cμ-Cδ intronic σδ region was used as acceptor site. Using polymerase chain reaction, we subsequently analyzed whether similar Sμ-σδ recombinations occur in normal tonsils containing IgM^?IgD⁺ plasma cells. Multiple products with a size range of 200–800 base pairs were detected in all four individuals, suggesting clustering of acceptor sites within σδ. Sequence analysis of three cloned products showed Sμ-σδ recombinations similar those observed in HCL. The σδ region contains a relatively high content of pentameric repeats with an extremely G-rich area and appears to function as a vestigial switch recombination site in normal and neoplastic IgM^?-IgD⁺ B cells.     

CD4<Superscript>+</Superscript> T Cells Downregulate Bcl-2 in Germinal Centers

Germinal centers (GCs) are the main site of T cell-dependent antibody responses. Upon antigen challenge, GCs comprise mostly B cells undergoing proliferation, somatic hypermutation and antigen-affinity selection. GC B cells down-modulate the expression of Bcl-2 protein and are highly sensitive to apoptosis to eliminate autoreactive or low-affinity cells. Bcl-2 is still expressed in a few GC cells, whose identity remains unclear. To address this issue, we examined by confocal microscopy the expression of Bcl-2 by different GC lymphocyte subsets in hyperplastic tonsils. We found that the vast majority of Bcl-2⁺ GC cells are T lymphocytes. Conversely, while in the mantle zone and in the interfollicular areas T cells are almost exclusively Bcl-2⁺, in the GC, most T lymphocytes are Bcl-2⁻. In addition, most of the CD4⁺ GC T cells are Bcl-2⁻, while nearly 100% of the CD8⁺ GC T cells are Bcl-2⁺. The Bcl-2 downregulation by both B and CD4⁺ T GC cells supports the concept that these two subsets may undergo a selection process in this microenvironment.     

Identification of a tonsil IgD+ B cell subset with phenotypical and functional characteristics of germinal center B cells

We have identified and isolated a subpopulation of IgD⁺ B cells (IgD⁺ CD38⁺ B cells) from human tonsils which expresses the germinal center (GC)-associated surface markers CD10, CD38, CD75, CD77 and CD95/Fas. The heterogeneity of expression of several markers on IgD⁺ CD38⁺ B cells suggests that this population can be further subdivided into two discrete subtypes. On a functional basis, IgD⁺ CD38⁺ B cells behave as GC B cells as they rapidly and spontaneously undergo apoptosis in vitro and cannot be stimulated to synthesize DNA upon cross-linking of the antigen receptor. However, in contrast with most GC B cells, IgD⁺ CD38⁺ B cells have not completed Ig class switching since they predominantly secrete IgM following stimulation in vitro and lack surface expression of secondary isotypes. Immunoenzymatic staining performed on tonsil tissue sections revealed that IgD⁺ CD38⁺ B cells are located in two distinct histological structures: within the GC of a few classical secondary follicles, in which they appear as scattered cells, and within rare atypical GC, homogeneously constituted of IgD⁺ B cells. Taken together, our findings indicate that IgD⁺ CD38⁺ B cells constitute a novel subset of GC B cells. The possibility that these cells could represent an early stage of the follicular reaction or be generated in response to certain bacterial carbohydrate antigens is discussed.     

Preliminary characterization and distribution of vicia villosa binding cells in human tonsils

Summary The lectin Vicia villosa (VV) has been used for the separation of human and murine contrasuppressor T cells. These cells were characterized in cryostat sections of human palatine tonsils by double staining with VV lectin and monoclonal antibodies to macrophages, lymphocytes and their subsets using a fluorescein-rhodamine technique. VV lectin had an affinity for the CD8⁺ subset of lymphocytes and for a subset of macrophages within the germinal centre. The number and distribution of VV lectin binding cells was studied in paraffin sections of formalin fixed tonsils by the avidin-biotin-peroxidase technique. Positive cells in the germinal centres, mantle, interfollicular zones and fibrous connective tissue septa were quantified using an image analyser. These were found in greatest density in the interfollicular zone, correlating with the known distribution of T cells in human palatine tonsils. The binding of VV lectin to a subset of macrophages appears not to have previously been described nor have VV lectin binding CD8⁺ lymphocytes been demonstrated in sections of human tissues.