Immunohistological analysis of tumour infiltrating lymphocytes in seminoma using monoclonal antibodies

Summary Immunological characterization of tumour infiltrating lymphocytes (TIL) by immunohistological techniques was carried out in 20 cases of stage I seminoma. Routine pathological examination of these surgical specimens showed typical seminoma in 20 cases. Eighteen cases showed obvious TIL and immunohistological staining on frozen specimens was performed in 12. TIL in seminomas were predominantly T-cells but B-cells were also identified. T-cells were distributed diffusely with predominance of the CD 8+ phenotype judged semiquantitatively. In contrast to the distribution of T-cells, B-cells tended to accumulate and occasionally formed lymphoid follicles. In such follicles the phenotypic pattern of B-cell antigens was comparable with secondary lymphoid follicles in lymphoid organs. There is an immunologically complex response to seminoma by the host with a predominant infiltration of cytotoxic/ suppressor T-cells and functional maturation of B-cells.     

Update on peripheral T-cell lymphomas with T-helper phenotype: Are there too many subtypes?

Follicular helper T (T_FH) cells are the providers of T-cell help to B-cells in the development of germinal centers and for the generation of most class-switched antibodies. The markers most commonly associated with T_FH activity are IL21, IL4, CD40L, BCL6, SAP, CXCR5/CXCL13, and ICOS. T-cell lymphoma genomic studies have shown that different T-cell lymphoma types express signatures typical for T_FH cells, this including angioimmunoblastic T-cell lymphoma (AITL), a related condition termed peripheral T-cell lymphoma with T_FH phenotype and primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder.Angioimmunoblastic T-cell lymphoma is a well-established entity, a clinically aggressive disease with a survival of 30% OS after 5 years. Molecular and clinical studies have confirmed this as a well-established clinicopathological entity with relatively specific gene mutations, including mutations found in hematopoietic precursor cells and others.Peripheral T-cell lymphoma with T_FH phenotype is an associated disorder with histology of PTCL but a T_FH phenotype, as defined by the expression of 2–3 immunohistochemical markers. Molecular studies on this entity are showing a partial overlap with AITL.Primary cutaneous CD4+ small/medium lymphoproliferative disorder is an entirely different process that takes place in the skin, showing frank cytologic atypia, monoclonal TCR rearrangement and T_FH phenotype in the context of a clinically benign lesion.Here we review the main clinical, molecular and diagnostic features of these three lymphoproliferative processes.     

The emerging role of T follicular helper (TFH) cells in aging: Influence on the immune frailty

The world population is undergoing a rapid expansion of older adults. Aging is associated with numerous changes that affect all organs and systems, including every component of the immune system. Immunosenescence is a multifaceted process characterized by poor response to vaccine and higher incidence of bacterial and viral infections, cancer, cardiovascular and autoimmune diseases. Immunosenescence has been associated with chronic low-grade inflammation referred to as inflammaging, whose underlying mechanisms remain incompletely elucidated, including age-related changes affecting components of the innate and adaptive immune system. T follicular helper (T_FH) cells, present in lymphoid organs and in peripheral blood, are specialized in providing cognate help to B cells and are required for the production of immunoglobulins. Several subsets of T_FH cells have been identified in humans and mice and modifications in T_FH cell phenotype and function progressively occur with age. Dysfunctional T_FH cells play a role in cancer, autoimmune and cardiovascular diseases, all conditions particularly prevalent in elderly subjects. A specialized population of Treg cells, named T follicular regulatory (T_FR) cells, present in lymphoid organs and in peripheral blood, exerts opposing roles to T_FH cells in regulating immunity. Indeed, changes in T_FH/T_FR cell ratio constitute a relevant feature of aging. Herein we discuss the cellular and molecular changes in both T_FH cells and T_FR cells that occur in aging and recent findings suggesting that T_FH cells and/or their subsets could be involved in atherosclerosis, cancer, and autoimmunity.     

Expression of CD137 (4-1BB) on human follicular dendritic cells

Follicular dendritic cells (FDCs) are the antigen (Ag)-trapping accessory cells of the germinal centres (GCs), essential for the development of humoral immune responses and memory. FDCs reside in the microenvironment of secondary lymphoid tissue where Ag-activated B cells expand, and undergo isotype switching and affinity maturation prior to becoming memory B cells. In addition to delivering Ag, FDCs also provide potent nonspecific accessory signals to the B cells, which are important for the GC reaction. In this report, we show that human tonsilar FDCs express the costimulatory molecule CD137. Surface expression of CD137 on FDCs was confirmed by immunofluorescent labelling and fluorescence-activated cell sorter analysis. CD137 was also highly expressed by the human cell line HK, which displays many characteristics of in vivo FDCs. The interaction between B cells and FDCs is essential for the GC reactions, and our finding suggests that CD137 plays a role in FDC-regulated B-cell responses.     

Roles of follicular helper and regulatory T cells in allergic diseases and allergen immunotherapy

Allergic diseases are characterized by overactive type 2 immune responses to allergens and immunoglobulin E (IgE)-mediated hypersensitivity. Emerging evidence suggests that follicular helper T (T_FH) cells, rather than type 2 T-helper (T_H2) cells, play a crucial role in controlling IgE production. However, follicular regulatory T (T_FR) cells, a specialized subset of regulatory T (T_REG) cells resident in B-cell follicles, restricts T_FH cell-mediated help in extrafollicular antibody production, germinal center (GC) formation, immunoglobulin affinity maturation, and long-lived, high-affinity plasma and memory B-cell differentiation. In mouse models of allergic asthma and food allergy, CXCR5⁺ T_FH cells, not CXCR5⁻ conventional T_H2 cells, are needed to support IgE production, otherwise exacerbated by CXCR5⁺ T_FR cell deletion. Upregulation of T_FH cell activities, including a skewing toward type 2 T_FH (T_FH2) and IL-13 producing T_FH (T_FH13) phenotypes, and defects in T_FR cells have been identified in patients with allergic diseases. Allergen immunotherapy (AIT) reinstates the balance between T_FH and T_FR cells in patients with allergic diseases, resulting in clinical benefits. Collectively, further understanding of T_FH and T_FR cells and their role in the immunopathogenesis of allergic diseases creates opportunities to develop novel therapeutic approaches.     

Abundance of follicular helper T cells in Peyer's patches is modulated by CD155

The secondary humoral immune response is characterized by plasma B cells secreting isotype‐switched and affinity‐matured antibodies. The efficient generation of plasma B cells in the GC depends on the presence of follicular helper T (T_FH) cells, a cell type thought to arise from naive CD4‐positive T cells by a hitherto unresolved differentiation pathway. Mice deficient for CD155, an adhesion receptor of the immunoglobulin superfamily, are impaired to mount a secondary humoral immune response upon oral administration of antigen, while the primary IgM response is unaffected. Here, we show that mice lacking CD155 harbor significantly reduced numbers of T_FH cells in their Peyer's patches. This was paralleled by a decreased frequency of T_FH cells in the GC. Moreover, the CD155 ligand CD226, which is involved in T‐cell activation, is down‐regulated during T_FH cell differentiation, resulting in a complete absence of CD226 on those T_FH cells residing in the GC. Concurrently, the expression of TIGIT/WUCAM, a newly discovered CD155 ligand, is induced in T_FH cells. Thus, these cells replace an activating by a putative inhibitory CD155‐binding partner during their differentiation.     

The hippo kinase MST1 negatively regulates the differentiation of follicular helper T cells

Follicular helper T (T_FH) cells are essential for inducing germinal centre (GC) reactions to mediate humoral adaptive immunity and antiviral effects, but the mechanisms of T_FH cell differentiation remain unclear. Here, we found that the hippo kinase MST1 is critical for T_FH cell differentiation, GC formation, and antibody production under steady-state conditions and viral infection. MST1 deficiency intrinsically enhanced T_FH cell differentiation and GC reactions in vivo and in vitro. Mechanistically, mTOR and HIF1α signalling is involved in glucose metabolism and increased glycolysis and decreased OXPHOS, which are critically required for MST1 deficiency-directed T_FH cell differentiation. Moreover, upregulated Foxo3 expression is critically responsible for T_FH cell differentiation induced by Mst1^−/−. Thus, our findings identify a previously unrecognized relationship between hippo kinase MST1 signalling and mTOR-HIF1α-metabolic reprogramming coupled with Foxo3 signalling in reprogramming T_FH cell differentiation.     

TFH cells in bystander and cognate interactions with B cells

Follicular T‐helper (T_FH) cells play a crucial role in three aspects of the germinal center (GC) response. They promote GC formation, arbitrate competition among GC B cells to determine the outcome of affinity maturation, and regulate GC output of memory and plasma cells to shape the long‐lived humoral immune memory. Of fundamental importance are dynamic physical interactions between T_FH and B cells, which are the main platform for T_FH cells to deliver “help” factors to B cells and also for reciprocal signaling from B cells to maintain the helper state of T_FH cells. Recent work has significantly expanded our understanding of how T‐B interactions are spatiotemporally regulated and molecularly orchestrated to fulfill those T_FH functions. In this review, we elaborate two modes of T‐B interactions, the antigen‐specific or cognate mode in which T_FH cells engage individual antigen‐presenting B cells and the antigen nonspecific bystander mode in which T_FH cells are engaged with the ensemble of follicular B cells. We discuss findings that indicate how short‐lived cognate T‐B contacts coupled with an intercellular positive feedback drive affinity‐based selection and how bystander interactions between T and B cells regulate follicular T‐cell recruitment and maintenance of an appropriate helper state. We argue that this combination of bystander and cognate interactions with B cells constantly shapes the internal state of T_FH cells and provides the platform to execute their helper functions.     

Human pregnancy levels of estrogen and progesterone contribute to humoral immunity by activating TFH/B cell axis

Circulating T_FH (cT_FH) cells express CXCR5, PD-1, and, when activated, ICOS, and release IL-21. According to the production of IFN-γ, IL-4, and IL-17 and expression of FoxP3, these cells are also classified as cT_FH1, cT_FH2, cT_FH17, and cT_FR cells, respectively. This CD4⁺T-cell subset is pivotal to efficient humoral immunity, and pregnancy appears to favor IgG production. Here, not only pregnancy amplified the in vivo production of anti-HBsAg IgG in HBV immunized women, but the frequency of cT_FH cells was directly correlated with estradiol levels. In vitro, pregnancy-related dose of 17-β-estradiol (E2) directly increased the percentage of different cT_FH subsets. While E2 and progesterone (P4) increased the proportion of differentiated T_FH cells derived from naïve CD4⁺T-cells, only E2 amplified the release of IL-21 in those cell cultures. In addition, E2 and P4 increased the proportion of memory B cells and plasma cells, respectively. In SEB-activated B/T_FH cell co-cultures, E2, in the presence of P4, increased the production of total IgG. Finally, among the hormones, P4 was stronger in upregulating the percentage of IL-10⁺T_FR cells. Collectively, our findings suggested that E2 and P4 cooperate in the humoral immune response by favoring the expansion of different cT_FH and B cell subsets.     

Langerhans cells promote early germinal center formation in response to Leishmania‐derived cutaneous antigens

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen‐primed CD4⁺ follicular helper T cells (T_FH). A tight and stable formation of T_FH/B cell conjugates is required for cytokine‐driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of T_FH/B cell conjugates is crucial for B‐cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for T_FH‐cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin⁺ DC subsets in vivo, we show that the functionality of T_FH/B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC‐depleted mice show a reduction in somatic hypermutation in B cells isolated from T_FH/B cell conjugates and markedly reduced GC reactions within skin‐draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B‐cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.     

Early protein synthesis in activated human peripheral blood mononuclear cells

Though B-cell division and Ig synthesis in response to pokeweed mitogen (PWM) require interaction with T-cells and monocytes, it is not clear which earlier events in B-cell activation share this requirement, and which are the result of direct interaction of mitogen with the B-cell. Having previously shown that the acceleration of lecithin synthesis in human B-cells at 16-20 hr requires both T-cells and monocytes, we now examine whether B-cells require similar interactions to increase their protein synthetic rate, another important activation event. At 21-24 hr of PWM stimulation, the stimulation index (SI) for incorporation of [35S]methionine into protein was 2.1 +/- 0.4 for unfractionated cells, 1.7 +/- 0.1 for B-cells, 2.5 +/- 0.1 for T-cells, and 3.4 +/- 0.5 for monocytes. Thus monocytes contributed substantially to early mitogen-induced protein synthesis by human peripheral blood mononuclear cells. When the monocyte/B-cell fraction (MB) and T-cell fraction (T) were mixed at various ratios in PWM-stimulated cultures, synergy was apparent at MB:T ratios of 1:1 and 1:2, indicating cell interactions augmented early mitogen-driven protein synthesis in at least one of these cell types. However, much or all of this synergy could be attributed to T-cells, whose protein synthetic response was augmented by B-cells and monocytes. In contrast, the early increase in B-cell protein synthesis appeared to be independent of cell interactions, since their SI of 1.7 was not influenced by varying the proportion of M- or T-cells over a 50-fold range. These contrasting results between two contemporary events fits the hypothesis that one (accelerated phospholipid synthesis) requires a first signal plus one or more cell interaction signals, whereas the other (accelerated protein synthesis) requires only the first signal.     

In vivo expression of interleukin-8, and regulated on activation, normal, T-cell expressed, and secreted, by human germinal centre B lymphocytes

T-cell homing within germinal centres (GCs) is required for humoral B-cell responses. However, the mechanisms implicated in the recruitment of T cells into the GC are not completely understood. Here we show, by immunohistology, and Northern and Western blots, that in vivo human GC B lymphocytes can express CxC and CC chemokines. Moreover, B-cell subset-specific experiments reveal that interleukin (IL)-8 and regulated on activation, normal, T-cell expressed, and secreted (RANTES) are predominantly expressed by GC centroblast and centrocytes, suggesting that chemokine expression is essential at stages in which B-lymphocytes engage in active antigen-dependent interactions with T lymphocytes. In keeping with this hypothesis, we show that the T cells recruited into the GC correlatively express the receptors for IL-8 and RANTES. We propose that chemokine expression is a key B-cell function that facilitates T-lymphocyte recruitment into the GCs and supports cognate B-cell : T-cell encounters. Moreover, our data are consistent with the impaired homing of T cells to secondary lymphoid organs in mice that are either deficient in CC and CxC chemokines or their receptors.     

Clonal composition and persistence of antigen-specific circulating T follicular helper cells

T follicular helper (T_FH) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4⁺ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC T_FH cells. However, the antigen specificity and relationship of these circulating T_FH (cT_FH) cells with other memory CD4⁺ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cT_FH and non-cT_FH subsets, although with different frequencies and effector functions. Interestingly, cT_FH and non-cT_FH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu-specific cT_FH and non-cT_FH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1⁺ cT_FH cells had a “GC T_FH-like” phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cT_FH and non-cT_FH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cT_FH with non-cT_FH cells and on the heterogeneity and persistence of antigen-specific human cT_FH cells.     

Syndromes mononucléosiques et pathologies hématologiques liés au virus d'Epstein-Barr

The Epstein-Barr virus (EBV), which preferentially infects B cells, persists in the infected subject as a latent asymptomatic infection. In adolescents, infectious mononucleosis is the symptomatic manifestation of primary EBV infection. The viral latency in the memory B-cells, the reservoir cells in peripheral blood in individuals is controlled by CD4 and CD8 positive T-cells. Immunodeficient patients are at high risk of developing EBV driven B-cell lymphomas as the consequence of the expression of oncogenic latency proteins LMP1 and EBNA2. These proteins expressed in infected B cells identify latency III or proliferation program in virus transformed B-cell, leading to lymphoid proliferation. In addition to immunodeficiency-related lymphomas, the most frequent lymphoid malignancies associated with EBV are the endemic Burkitt lymphoma, Hodgkin lymphoma and nasal type T-cell lymphoma.     

Interaction between dendritic cells and nerve fibres in lymphoid organs after oral scrapie exposure

In transmissible spongiform encephalopathies (TSEs), the infectious agent, called PrPsc, an abnormal isoform of the cellular prion protein, accumulates and replicates in lymphoid organs before affecting the nervous system. To clarify the cellular requirements for the neuroinvasion of the scrapie agent from the lymphoid organs to the central nervous system, we have studied, by confocal microscopy, the innervations within Peyer’s patches, mesenteric lymph nodes and the spleen of mice in physiological conditions and after oral exposure to prion. Contacts between nerve fibres and PrPsc-associated cells, dendritic cells (DCs) and follicular dendritic cells (FDCs), were evaluated in preclinical prion-infected mice. Using a double immunolabelling strategy, we demonstrated the lack of innervation of PrPsc-accumulating cells (FDCs). Contacts between nerve fibers and PrPsc-propagating cells (DCs) were detected in T-cell zones and cell-trafficking areas. This supports, for the first time, the possible implication of dendritic cells in the prion neuroinvasion process.