Tonsil Germinal Center Research Articles

Surgical outcomes and unique histological features of tonsils after tonsillectomy in adults with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome

ObjectivesData on the adult-onset periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome are scarce. European studies reported that unlike pediatric-onset PFAPA, tonsillectomy is ineffective for adult-onset PFAPA. The aims of this study were (1) to assess the response to tonsillectomy in a cohort of Japanese adult-onset PFAPA patients and (2) to evaluate the histologic appearance of tonsils in adult-onset PFAPA patients and to compare them with those of tonsils from age- and sex-matched controls with chronic tonsillitis. MethodsIn this retrospective cohort study, 5 adults with PFAPA and 15 controls who had undergone tonsillectomy were recruited. The size of the tonsil germinal centers was measured by hematoxylin and eosin staining, and the number and density of B and T lymphocytes in germinal centers were measured by immunohistochemistry, using CD3, CD4 and CD8 as T cell markers and CD20 as B cell marker. ResultsAll patients had complete remission of the symptoms after surgery. PFAPA patients had significantly smaller germinal center areas than controls. The number and density of CD8+ cells in germinal centers were significantly lower in tonsils from PFAPA compared with controls. No differences were found between the two groups in CD3+, CD4+, and CD20+ cells. These results are compatible with the tonsillar features of pediatric-onset PFAPA. ConclusionOur report demonstrates that tonsillectomy might be effective for adult-onset PFAPA and that tonsils of adult- and pediatric-onset PFAPA share the same histological features. These results suggest that the pathogenic mechanisms of adult- and pediatric-onset PFAPA are identical.

Abstract 696: Patterns of TIGIT expression in normal lymphatic tissue, inflammation and cancer

Abstract T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. To study patterns of TIGIT expression, “microenvironment tissue microarrays” containing 4mm tissue spots were constructed from lymph nodes of healthy (n=3) and HIV infected individuals (n=2), normal tonsils (n=3), Hashimoto thyroiditis (n=10), sarcoidosis (n=10), lichen sclerosus (n=2), IgG4-pancreatitis (n=2), rheumatoid arthritis (n=2) colorectal (n=5) and lung cancers (n=2) and analyzed for expression of TIGIT, PD-1 and standard lymphocyte markers by brightfield and multiplex fluorescence immunohistochemistry. TIGIT expression was seen in CD8+ cytotoxic T cells, CD4+ T-helper cells, FOXP3+ regulatory T cells and in NK cells, but not in CD11c+ dendritic cells, CD68+ macrophages and CD20+ B lymphocytes. TIGIT expression paralleled that of PD-1. More than 70% of TIGIT positive cells were PD-1 positive and more than 90% of the PD-1 positive cells were TIGIT positive. Expression of both proteins varied between different tissue compartments. TIGIT expression in tonsil gradually increased from the interfollicular area over the marginal/mantle zone to the germinal center in all T cell subtypes. Stronger expression of TIGIT and PD-1 than in tonsil germinal centers (2-3 fold) was only found in Hashimoto thyroiditis germinal centers. Marked location specific variations were also seen in other inflammatory diseases and in cancers. In general, TIGIT and PD-1 expression was higher in lymphocyte-dense compartments, such as areas of lymphocytic infiltration in sarcoidosis, IgG4 pancreatitis or rheumatoid arthritis, than in areas containing fewer and scattered lymphocytes. In lung and colorectal cancers, the density of TIGIT and PD-1 expressing T cells was highest at the invasion front. Also, the TIGIT and PD-1 expression levels were typically stronger in tumor adjacent stromal CD8+ cells than in tumor infiltrating CD8+ cells. In conclusion, TIGIT is regularly expressed in a large subset of T cells. The variable expression levels of TIGIT and PD-1 between cell types and tissue compartments illustrates the high complexity of immune response. The frequent co-expression of TIGIT and PD-1 may offer an opportunity for co-targeting these proteins with checkpoint inhibitor drugs. Citation Format: Niclas C. Blessin, Ronald Simon, Martina Kluth, Kristine Fischer, Claudia Hube-Magg, Wenchao Li, Georgia Makrypidi-Fraune, Björn Wellge, Tim Mandelkow, Nicolaus F. Debatin, Guido Sauter, Waldemar Wilczak, Andrea Hinsch. Patterns of TIGIT expression in normal lymphatic tissue, inflammation and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 696.

Bcl-2 as a Therapeutic Target in Human Tubulointerstitial Inflammation.

In lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ adaptive immune cell networks that amplify local tissue damage. Since conventional therapy appears ineffective for severe TII, and these patients often progress to renal failure, understanding in situ mechanisms might reveal new therapeutic targets. This study was undertaken to assess whether dysregulated apoptotic regulators maintain local adaptive immunity and drive inflammation in TII. This study utilized novel computational approaches that, when applied to multicolor confocal images, quantified apoptotic regulator protein expression in selected lymphocyte subsets. This approach was validated using laser-capture microdissection (LCM) coupled to quantitative polymerase chain reaction (qPCR). Furthermore, the consequences of dysregulated apoptotic mediator expression were explored in a murine model of lupus nephritis. Analyses of renal biopsy tissue from patients with lupus nephritis and those with mixed cellular renal allograft rejection revealed that the B cell lymphoma 2 protein (Bcl-2) was frequently expressed in infiltrating lymphocytes, whereas expression of myeloid cell leukemia 1 was low. In contrast, the reciprocal pattern of expression was observed in tonsil germinal centers. These results were consistent with RNA expression data obtained using LCM and qPCR. Bcl-2 was also highly expressed in tubulointerstitial infiltrates in (NZB × NZW)F1 (NZB/NZW) mice. Furthermore, treatment of NZB/NZW mice with ABT-199, a selective oral inhibitor of Bcl-2, prolonged survival and prevented proteinuria and development of TII in a lupus prevention model. Interestingly, glomerular immune complexes were partially ameliorated by ABT-199 treatment, and serum anti-double-stranded DNA antibody titers were unaffected. These data demonstrate that Bcl-2 is an attractive therapeutic target in patients with lupus nephritis who manifest TII.

IL-17 superfamily cytokines modulate normal germinal center B cell migration.

The germinal center (GC) is a dynamic structure formed by proliferating B cells in the follicles of secondary lymphoid organs during T cell-dependent antibody responses to exogenous antigens. GC is composed by a dark zone, enriched in proliferating centroblasts (CBs), and a light zone where CBs migrate and transform into centrocytes (CCs), a minority of which is selected to survive, undergoes Ig class-switch recombination, and differentiates into memory B cells or long-lived plasma cells. CBs express CXCR4 and are attracted to the dark zone by stromal cell-derived CXCL12, whereas CCs express CXCR5 and are recruited to the light zone along a gradient of CXCL13 produced by follicular dendritic cells (FDCs). Therefore, CXCL12 and CXCL13 play crucial roles in the regulation of GC B cell trafficking. Among the numerous molecules involved in GC formation, IL-17A represents a recent addition. Its involvement has been demonstrated in mouse models of human autoimmune or infectious diseases. IL-17A belongs to the IL-17 cytokine superfamily, together with 5 additional structurally related cytokines. We have recently demonstrated that IL-17A renders freshly isolated tonsil GC B cells competent to migrate to CXCL12 and CXCL13 through a NF-κBp65-dependent mechanism. Here, we review the role of IL-17A on GC cells and discuss, for the first time, common effects of the cognate cytokines IL-25 and IL-17B on GC B cell function.

Interleukin-17A promotes the growth of human germinal center derived non-Hodgkin B cell lymphoma

Interleukin (IL)-17A belongs to IL-17 superfamily and binds the heterodimeric IL-17 receptor (R)(IL-17RA/IL-17RC). IL-17A promotes germinal center (GC) formation in mouse models of autoimmune or infectious diseases, but the role of IL-17A/IL-17AR complex in human neoplastic GC is unknown. In this study, we investigated expression and function of IL-17A/IL-17AR in the microenvironments of 44 B cell non-Hodgkin lymphomas (B-NHL) of GC origin (15 follicular lymphomas, 17 diffuse large B cells lymphomas and 12 Burkitt lymphomas) and 12 human tonsil GC. Furthermore, we investigated the role of IL-17A in two in vivo models of GC B cell lymphoma, generated by s.c. injection of SU-DHL-4 and OCI-Ly8 cell lines in Severe combined immunodeficiency (SCID)/Non Obese Diabetic (NOD) mice. We found that: (i) B-NHL cell fractions and tonsil GC B cells expressed IL-17RA/IL-17RC, (ii) IL-17A signaled in both cell types through NF-kBp65, but not p38, ERK-1/2, Akt or NF-kBp50/105, phosphorylation, (iii) IL-17A was expressed in T cells and mast cells from neoplastic and normal GC microenvironments, (iv) IL-17A rendered tonsil GC B cells competent to migrate to CXCL12 and CXCL13 by downregulating RGS16 expression; (v) IL-17A stimulated in vitro proliferation of primary B-NHL cells; (vi) IL-17A (1 μg/mouse-per dose) stimulated B-NHL growth in two in vivo models by enhancing tumor cell proliferation and neo-angiogenesis. This latter effect depended on IL-17A-mediated induction of pro-angiogenic gene expression in tumor cells and direct stimulation of endothelial cells. These data define a previously unrecognized role of human IL-17A in promoting growth of GC-derived B-NHL and modulating normal GC B cell trafficking.

A T cell-dependent mechanism for the induction of human mucosal homing IgA-secreting plasmablasts (39.5)

Abstract Mucosal IgA secreted by local plasma cells (PCs) is a critical component of mucosal immunity. Although IgA class switching can occur at mucosal sites in a T cell-independent manner, high-affinity PCs are optimally generated in germinal centers (GCs) in a T cell-dependent fashion. However, the mechanism of how CD4+ helper T cells induce mucosal-homing IgA-PCs remains unclear. We show here that TGFb1 and IL-21, produced by follicular helper T cells (TFH), synergize to generate abundant IgA-plasmablasts (PBs) that express CCR10. In the presence of IL-21, TGFb1 promotes naive B cell proliferation and differentiation. In addition it overrides IL-21-induced IgG class switching in favor of IgA. Furthermore, in combination with IL-21, TGFb1 downregulates CXCR5, while upregulating CCR10 on PBs, enabling their exit from GCs and migration towards local mucosa. This is supported by the presence of CCR10+IgA+ cells in tonsil GCs. In conclusion, TFH, through IL-21 and TGFb1 contribute to the generation of human mucosal-homing IgA+ PCs. Thus, mucosal vaccines should aim to induce robust TFH responses.

Ectopic Germinal Centers Are Rare in Sjögren’s Syndrome Salivary Glands and Do Not Exclude Autoreactive B Cells

This study reports on the characterization of B cells of germinal center (GC)-like structures infiltrating the salivary glands (SGs) of patients with Sjögren's syndrome. Eight two-color combinations were devised to characterize the phenotype of these B cells in 11 SG specimens selected from biopsies obtained from 40 Sjögren's syndrome patients and three normal tonsils. The 9G4 mAb, which recognizes V4.34-encoded autoAbs, enabled us to identify autoreactive B cells. Quantitative RT-PCR was used to determine the level of mRNAs for activation-induced cytidine deaminase (AICDA), repressors and transcription factors. CD20(+)IgD(-)CD38(+)CD21(+)CD24(-) B cells, similar to those identified in tonsil GCs, were seen in the SGs of four patients and, and since they expressed AICDA, they were termed "real GCs". CD20(+)IgD(+)CD38(-)CD21(+)CD24(+) B cells, seen in aggregates from the remaining seven samples, were characteristically type 2 transitional B cells and marginal zone-type B cells. They lacked AICDA mRNAs and were termed "aggregates". Real GCs from SGs contained mRNAs for Pax-5 and Bcl-6, like tonsil GC cells, whereas aggregates contained mRNAs for Notch-2, Blimp-1, IRF-4, and BR3, similar to marginal zone B cells. Further experimental data in support of this dichotomy included the restriction of CXCR5 expression to real GC cells, while sphingosine 1-phosphate receptor 1 was expressed only in aggregates. In contrast, both types of B cell clusters expressed the idiotype recognized by the 9G4 mAb. Our data indicate that, in SGs, a minority of B cell clusters represent genuine GC cells, while the majority manifest features of being type 2 transitional B cells and marginal zone cells. Interestingly, both types of B cell aggregates include autoreactive B cells.

A T Cell-Dependent Mechanism for the Induction of Human Mucosal Homing Immunoglobulin A-Secreting Plasmablasts

Mucosal immunoglobulin A (IgA) secreted by local plasma cells (PCs) is a critical component of mucosal immunity. Although IgA class switching can occur at mucosal sites, high-affinity PCs are optimally generated in germinal centers (GCs) in a T cell-dependent fashion. However, how CD4(+) helper T cells induce mucosal-homing IgA-PCs remains unclear. Here, we show that transforming growth factor beta1 (TGFbeta1) and interleukin 21 (IL-21), produced by follicular helper T cells (Tfh), synergized to generate abundant IgA-plasmablasts (PBs). In the presence of IL-21, TGFbeta1 promoted naive B cell proliferation and differentiation and overrode IL-21-induced IgG class switching in favor of IgA. Furthermore, TGFbeta1 and IL-21 downregulated CXCR5 while upregulating CCR10 on plasmablasts, enabling their exit from GCs and migration toward local mucosa. This was supported by the presence of CCR10(+)IgA(+)PBs in tonsil GCs. These findings show that Tfh contribute to mucosal IgA. Thus, mucosal vaccines should aim to induce robust Tfh responses.

Small-Molecule XIAP Antagonist Restores Caspase-9-Mediated Apoptosis in XIAP-Positive Diffuse Large B-Cell Lymphoma Cells.

Clinical outcome in patients with diffuse large B-cell lymphomas (DLBCL) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including XIAP. XIAP suppresses apoptosis through inhibiting active caspases-3, -7 and -9. In this study we investigated if the small-molecule XIAP antagonist 1396–12 induces cell death in cultured lymphoma cells of DLBCL patients and whether it is possible to predict whether a DLBCL will be sensitive to the XIAP antagonist. Treatment with this XIAP antagonist resulted in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy refractory and responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal center B-cells from healthy donors. XIAP antagonist-sensitive cases were characterized by high expression levels of XIAP and relatively low expression levels of Bcl-2. In addition, we found that XIAP antagonist sensitive lymphomas are characterized by constitutive caspase-9 activation and that the apoptosis inducing effect of the XIAP antagonist depends on this constitutive caspase 9 activity. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in DLBCL cells by restoring caspase 9 mediated apoptosis and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers suggesting the possibility of pre-defining patients most likely to benefit from XIAP antagonist therapy.

Kinetics of Human B Cell Behavior and Amplification of Proliferative Responses following Stimulation with IL-21

Although recent studies indicated that IL-21 is an important regulator of human B cell activation, detailed comparison of the effects of IL-21 on distinct B cell subsets have not been performed. Our studies revealed that IL-21R is expressed by naive and germinal center B cells, but not memory or plasma cells. IL-21R was increased on naive and memory B cells following in vitro activation. Investigation into the kinetics and magnitude of responses of human B cells to IL-21 revealed that IL-21 potently augmented proliferation of CD40L-stimulated neonatal, splenic naive, and memory and tonsil germinal center B cells. This response exceeded that induced by IL-4, IL-10, and IL-13, cytokines that also induce B cell proliferation. Remarkably, CD40L/IL-21-stimulated naive B cells underwent the same number of divisions as memory cells and exhibited a greater enhancement in their response compared with CD40L alone than memory B cells. Therefore, IL-21 is a powerful growth factor for naive B cells. This may result from the higher expression of IL-21R on naive, compared with memory, B cells. Stimulation of human B cells with CD40L/IL-21 also induced IL-10 production and activation of STAT3. We propose that IL-21 may have therapeutic application in conditions of immunodeficiency where it could expand naive B cells, the predominant B cell subset in such patients. Conversely, because IL-21 is increased in murine models of lupus, dysregulated IL-21 production may contribute to perturbed B cell homeostasis observed in systemic lupus erythematosus. Thus, antagonizing IL-21 may be a novel strategy for treating Ab-mediated autoimmune diseases.

HIV-specific effector cytotoxic T lymphocytes and HIV-producing cells colocalize in white pulps and germinal centers from infected patients

AbstractHuman immunodeficiency virus (HIV) infection is characterized by the massive infiltration of secondary lymphoid organs with activated CD8+ T lymphocytes. While converging data indicated that these cells were HIV-specific cytotoxic T lymphocytes (CTLs) responsible for HIV spread limitation, direct evidence was lacking. Here, the presence of HIV-specific effector CTLs was demonstrated directly ex vivo in 15 of 24 microdissected splenic white pulps from an untreated patient and in 1 of 24 tonsil germinal centers from a second patient with incomplete viral suppression following bitherapy. These patients had plasma HIV RNA loads of 5900 and 820 copies per milliliter. The frequencies of HIV-1 DNA+ cells in their lymphoid organs were more than 1 in 50 and 1 in 175, respectively. Spliced viral messenger RNA (a marker for ongoing viral replication) was present in most immunocompetent structures tested. Conversely, CTL activity was not found in spleens from 2 patients under highly active antiretroviral therapy, with undetectable plasma viral load. These patients had much lower spleen DNA+ cell frequencies (1 in 2700 and 1 in 3800) and no white pulps containing spliced RNA. CTL effector activity as well as spliced viral messenger RNA were both concentrated in the white pulps and germinal centers. This colocalization indicates that viral replication in immunocompetent structures of secondary lymphoid organs triggers anti-HIV effector CTLs to these particular locations, providing clues to target therapeutic intervention.

Up-regulation of telomerase in human B lymphocytes occurs independently of cellular proliferation and with expression of the telomerase catalytic subunit.

Telomerase activity is up-regulated 1000-fold higher in human tonsil germinal center B cells compared to resting naive or memory B cells, and telomerase expression can be re-activated in vitro resting B cells. To understand the mechanism(s) of telomerase regulation, quiescent B cell from peripheral blood or tonsil were activated with different combinations of various stimuli. Cross-linking surface (s)IgD or sIgM of B cells induced marked up-regulation of telomerase enzymatic activity in the absence of cellular proliferation. Low level cross-linkage of surface molecules by soluble anti-IgM did not up-regulate the telomerase activity. However, the inability of soluble anti-IgM to up-regulate the telomerase activity was corrected by additional signals from soluble anti-CD40 antibody engagement or IL-4 / IL-10. Activation of B cell proliferation with Epstein-Barr virus failed to up-regulate telomerase, further suggesting that up-regulation of telomerase is an event independent of B cell proliferation. Telomerase induction occurred in the late G1 phase of the cell cycle and did not require entry into S phase. Up-regulation of telomerase enzymatic activity correlated primarily with the induction of expression of the hTERT gene, the catalytic subunit to telomerase, suggesting that control of telomerase regulation resides at the level of the catalytic subunit of this holoenzyme.

Surface Molecule Loss and Bleb Formation by Human Germinal Center B Cells Undergoing Apoptosis: Role of Apoptotic Blebs in Monocyte Chemotaxis

Human tonsil germinal center (GC) B cells rapidly undergo apoptosis in culture. Annexin-V binding shows an early event in this process. In the present study, this method has been used to label apoptotic GC B cells and to analyze additional surface molecules. The expression of all of the molecules studied was reduced in apoptotic (annexin-V(+)) GC B cells, and the reduction was more marked for CD11a, CD21, CD22, CD49d, and CD54, molecules that participate in survival interaction for GC B cells. The analysis of CD54, one of the molecules that was more drastically reduced, showed that GC, but not mantle zone, B cells actively secrete CD54 to the culture supernatant (SN). The secreted CD54 was partly released from the GC B cells in a particulate form as demonstrated by centrifugation. Further experiments using filtration, fluorescence microscopy, electron microscopy, and flow cytometry analysis showed that GC B cells released to the culture SN a population of spherical membranous vesicles of about 0.18 micrometers in size, similar to the blebs described in other apoptosis systems. Bleb formation depended on active metabolism, Ca(2+), and, in part, on microfilament integrity. GC B-cell-derived blebs were clearly associated with apoptosis, as antiapoptotic stimuli prevented their formation. In addition, GC B-cell-derived blebs contained the adhesion molecules previously studied. Consequently, bleb formation might contribute to the surface molecule loss occurring in apoptotic GC B cells. Finally, a chemotaxis assay showed that GC B-cell blebs were chemotactic for human monocytes, suggesting that this mechanism might operate in vivo.

Telomerase is up-regulated in human germinal center B cells in vivo and can be re-expressed in memory B cells activated in vitro.

The extensive proliferation that B lymphocytes undergo in germinal centers could compromise generation of long term B cell memory if there occurs shortening of the telomeres of germinal center B cells with cell division. Telomere length, which is thought to act as a "mitotic clock" for somatic cells that dictates cellular senescence, can be preserved by the enzyme telomerase. Human tonsil germinal center B cells consistently expressed 100- to 1000-fold higher levels of telomerase activity than naive or memory B cells, which had no or very low detectable activity, as analyzed by the telomere repeat amplification protocol assay. In vitro stimulation of human memory B cells through surface Ig or CD40 was capable of up-regulating telomerase. The findings suggest that longevity of B cell memory is maintained, despite multiple cell divisions in the generation of a memory B cell, by up-regulation of telomerase in germinal center and activated memory B cells.

Dendritic cells in the bursal follicles and germinal centers of the chicken's caecal tonsil express vimentin but not desmin.

Immunohistochemical studies with anti-vimentin and anti-desmin monoclonal antibodies were designed to determine the origin of bursal secretory dendritic cells (SDC) and follicular dendritic cells. The binding sites of anti-vimentin, anti-desmin, and anti-chicken-IgG specific monoclonal antibodies were visualized with a biotinylated anti-mouse-IgG, ABC Elite kit, and 4-chloronaphthol. Cells were double stained (anti-vimentin and rabbit anti-chicken-IgG Fc) to determine if the vimentin positive cells possessed surface IgG. Vimentin positive cells were observed in the cortex and medulla of the bursa and germinal center and lymphoepithelial compartment of the caecal tonsil. The mesenchymal reticular cell, the basic supporting cell of the germinal center, was stained prominently by anti-vimentin and anti-desmin. Both antibodies stained the bursal cortex but only anti-vimentin bound the bursal secretory dendritic cell of the medulla. In addition to being vimentin positive and desmin negative, the bursal secretory dendritic cell possessed and the follicular dendritic cell appeared to possess IgG on their surfaces. In all the observations, B-cells were vimentin negative. These studies suggest that follicular dendritic cells and mesenchymal reticular cells in the caecal tonsil's germinal centers may be functionally different cell populations while the bursal secretory dendritic cell and follicular dendritic cell of the caecal tonsil may have a common origin.

Isolation and characterization of Leu 7+ germinal-center cells with the T helper-cell phenotype and granular lymphocyte morphology.

Leu 7+ cells in germinal centers of lymphoid tissues largely (greater than 90%) coexpress the T helper-cell marker, Leu 3. In this study we have isolated Leu 7+ (Leu 3+) cells from pharyngeal and palatine tonsils and we have analyzed their surface phenotype, morphologic and cytochemical characteristics, and functional properties. All of these features have been compared with those of T helper-cell populations with natural killer (NK)-like characteristics that we have previously described in peripheral blood. Leu 7+ (Leu 3+) cells from tonsil germinal centers display morphological and cytochemical features of granular lymphocytes and express the T3 marker in the absence of Leu 15. Following stimulation with phytohemagglutinin (PHA) or anti-T3, Leu 7+ (Leu 3+) cells express interleukin-2 (IL-2) receptors and proliferate to some extent in response to IL-2. The localization of Leu 7+ (Leu 3+) cells in B-dependent areas of lymphoid tissues suggests that they may play a regulatory role in B-cell proliferation and/or differentiation. Here we show that Leu 7+ (Leu 3+) cells do not produce B-cell growth factor (BCGF) and do not help pokeweed mitogen (PWM)-driven B-cell differentiation. Therefore, Leu 7+ (Leu 3+) germinal-center cells are distinct from "classic" T-helper cells of blood and lymphoid tissues.