Helper T cell-regulated B cell immunity.

In this review, we will discuss the cascade of cellular and molecular events in the immune response to protein antigens that regulate the development of high-affinity B cell memory. The behavior of antigen-experienced pMHCII+ dendritic cells DCs and the dynamics of their interaction with specific T-helper (Th) cells define the first developmental checkpoint for adaptive immunity in vivo. Recent studies provide insight into the basis of Th cell clonal selection and the requirements and consequences of antigen priming in this responsive Th cell compartment. Antigen-specific Th cells expand to become the cognate regulators of effector B cell responses and initiators of the germinal center reaction and memory B cell development. We will discuss the development and role of these diverse mixtures of antigen-specific B cells in the control of B cell memory and long-term humoral immunity that underpin effective protein vaccination.

CD4 promotes breadth in the TCR repertoire.

A diverse population of MHC class II-restricted CD4 lineage T cells develops in mice that lack expression of the CD4 molecule. In this study, we show that the TCR repertoire selected in the absence of CD4 is distinct, but still overlapping in its properties with that selected in the presence of CD4. Immunization of mice lacking CD4 caused the clonal expansion of T cells that showed less breadth in the range of Ag-binding properties exhibited by their TCRs. Specifically, the CD4-deficient Ag-specific TCR repertoire was depleted of TCRs that demonstrated low-affinity binding to their ligands. The data thus suggest a key role for CD4 in broadening the TCR repertoire by potentiating productive TCR signaling and clonal expansion in response to the engagement of low-affinity antigenic ligands.

Priming by microbial antigens from the intestinal flora determines the ability of CD4+ T cells to rapidly secrete IL-4 in BALB/c mice infected with Leishmania major.

Infection of BALB/c mice with Leishmania major results in the rapid accumulation of IL-4 transcripts within CD4(+) T cells that react to the parasite Leishmania homologue of mammalian RACK1 (LACK) Ag. Because memory/effector cells secrete IL-4 more rapidly than naive cells, we sought to analyze the phenotype of these lymphocytes before infection. Indeed, a fraction of LACK-specific CD4(+) T cells expressed a typical CD62 ligand(low)CD44(high)CD45RB(low) phenotype in uninfected mice. LACK-specific T cells were primed in gut-associated lymphoid tissues by cross-reactive microbial Ags as demonstrated by their reactivity with bacterial extracts and by the ability of APCs from the mesenteric LN of BALB/c mice to induce their proliferation. Also, mice in which the digestive tract has been decontaminated exhibited a reduced proportion of LACK-specific T cells expressing a memory/effector phenotype and did not exhibit the early accumulation of IL-4 transcripts induced by L. major. Thus, LACK-specific T cells represent a subset of CD4(+) T cells which have acquired the ability to rapidly secrete IL-4 as the result of their priming by cross-reactive microbial Ags. Tracking the fate of these cells may provide information about the regulation of cell-mediated immune responses to gut Ags in physiological and pathological situations.

Selective activation and expansion of high-affinity CD4+ T cells in resistant mice upon infection with Leishmania major.

Using multimers of MHC class II molecules linked to a peptide derived from the Leishmania LACK antigen, we have compared the fate of parasite-specific CD4+ T cells in resistant and susceptible mice transgenic for the beta chain of a LACK-specific TCR. Activated T cells were readily detected in the draining lymph nodes of infected animals. Although the kinetics of activation and expansion were similar in both strains, T cells from susceptible and resistant mice expressed low- and high-affinity TCR, respectively. As T cells from resistant mice produced more IFN-gamma and less IL-4 than those from susceptible animals, our results suggest that differences in TCR usage between MHC-matched animals may influence the development of the antiparasite immune response.

Immunological tolerance to a pancreatic antigen as a result of local expression of TNFalpha by islet beta cells.

Recent experiments have suggested that tumor necrosis factor alpha (TNFalpha) can down-regulate islet-specific T cells and prevent the development of autoimmune diabetes. Here we demonstrate that transgenic mice expressing both TNFalpha and the Leishmania major LACK antigen in the pancreas (RIP-TNFalpha/RIP-LACK) exhibit an impaired ability to mount a CD4+ T cell response against LACK. In addition, peripheral CD4+ T cells from TCR transgenic mice (TCR-LACK/RIP-TNFalpha/RIP-LACK) produced reduced interleukin-2 but elevated levels of T helper 2 cytokines in response to LACK peptide in vitro. Taken together, our data suggest that TNFalpha may act in vivo to modulate a potentially damaging self-reactive T cell response by inducing tolerance to pancreatic antigens.

Actin content and organization of microfilaments in primary cultures of mouse embryonic fibroblasts (in vitro ageing).

Actin distribution in serially passaged embryonic mouse fibroblasts has been visualized by the anti-actin-PAP method; the organization of the microfilaments has been observed by electron microscopy (SEM and TEM). Four successive actin patterns have been identified: early (few well-organized bundles of microfilaments), middle-aged (many well-organized bundles and patches around the nucleus), late (numerous ill-organized filamentous structures and diffuse perinuclear-actin) and "senescent" (heavy packs of short microfilaments around the nucleus). All the observed actin-positive filaments were disrupted by cytochalasin B treatment. The cytoplasmic actin complex was cell-age and not cell-size-dependent; it behaved differently from the cytoplasmic microtubular complex to serially subcultivated fibroblasts. Measurements of the cell-protein content (Lowry's method) and SDS-polyacrylamide gel electrophoresis (Laemmli's method) have been performed in the successive population doubling levels (PDL) of the primary cultures. Triton-insoluble actin increased in parallel with total protein and reached about 4% of the total proteins in all the PDLs. Triton-soluble actin also increase at the beginning of the middle-aged period (generally 6 PDL) and another in declining cultures (generally 10 PDL). Total actin amounted to about 8% of the total proteins in early fibroblasts, to about 16% at the beginning of the middle-aged period and to about 20% in the declining terminal cultures. Taking into account all the known characteristics of subcultivated primary cultures, we tentatively consider the evolution of the fibroblasts as an in vitro differentiation followed by true in vitro senescence in the declining cultures. Regarding the cytoplasmic actin-complex, senescence would be characterized by a sharp increase in soluble actin, an unbalanced ratio between soluble and insoluble actin and an impairment of the ability of the microfilaments to form well-organized bundles.

Relationships between cytoplasmic microtubular complex, DNA synthesis and cell morphology in mouse embryonic fibroblasts (effects of age, serum deprivation, aphidicolin, cytochalasin B and colchicine).

Aging, aphidicolin, serum deprivation and cytochalasin B induce a decrease in the rate of DNA synthesis, an increase in cell flattening (cell surface increase) and an extension of the cytoplasmic microtubular complex (CMTC). Age and experimental conditions affect the protein content of the cell, but there is no relationship between cell morphology and cell protein content. Serum deprivation, aphidicolin and cytochalasin B are more effective on DNA synthesis and cytoplasmic actin complex (CAC) of late than of early fibroblasts. Despite these facts, the cell morphology of late cells is fairly stable and is not affected by experimental conditions, which exert an "aging effect" upon the cell morphology in earlier cultures. Colchicine acts upon the CMTC, cell morphology and DNA synthesis at all ages of the cultures. It also induces disruption of the CAC, the intensity of the disruption depending on both the length of the treatment and the age of the culture: the sensitivity of the actin-microfilaments to colchicine increases with the mitotic age of the cells. We suggest that the microtubular integrity is needed, but not sufficient, to preserve the organization of the CAC into microfilaments. We propose a logical model comprising feedback loops between the number of the mitotic cycles, the rate of DNA synthesis, the extention rate of the plasma membranes and CMTC in normal fibroblasts. CMTC is associated, in this model, with the expression of negative or positive controls, depending on the grade of its extension (Fig. 9).