Human monoclonal Fab fragments specific for viral antigens from combinatorial IgA libraries.

BACKGROUND: IgA constitutes the first line of immune defense, interacting with a variety of environmental antigens. Following infection with respiratory syncytial virus (RSV) individuals frequently exhibit elevated serum IgA titers specific for the virus. Previously combinatorial IgG libraries have successfully been used to clone such human antibody responses. OBJECTIVES: Here we evaluate the possibility of constructing combinatorial IgA libraries on the surface of filamentous phage to retrieve human viral-specific IgA Fab fragments. STUDY DESIGN: Bone marrow from an HIV-1 seropositive donor was used as RNA source to construct combinatorial IgA kappa and lambda libraries of approximately 10(7) clones. RESULTS: By affinity selection using an immobilized recombinant RSV FG protein, two unique IgA Fab fragments producing clones (AD5 and AD23) reactive with the selecting antigen were isolated. One of the Fab fragments was found to be specific for RSV F glycoprotein and bind with high apparent affinity (2 x 10(8) M-1). The other binds with lower affinity and exhibits cross-reactivity with other antigens. CONCLUSION: The strategy described, involving construction of combinatorial IgA libraries on the surface of filamentous phage, should be generally applicable to the investigation of both mucosal and systemic human IgA immune responses, and may become an important tool for evaluation of mucosal vaccine regimes.

Immunoregulation by cytokines.

The study of cytokine biology relevant to the in vivo (patho)physiology of the immune response is complicated by a series of features inherent to the cytokine system. The present survey focuses on the mechanisms governing the function of cytokines that may give rise to methodological and conceptual problems concerning in vivo manipulations of immunologically relevant cytokines. Special emphasis is laid on the complex interrelation between individual cytokines (cascades, synergy, anergy, pleiotropism, and redundancy), as well as on the mechanisms that guarantee a compartmentalization of cytokines, i.e., a chronological, spatial, cell-type differentiation stage, and activation-dependent restriction of their function. The in vivo effects of cytokines can be studied either by augmenting their concentration or reducing their bioavailability. The advantages of local and systemic cytokine injections, usage of transgenes, or expression as gene products encoded by recombinant viruses are discussed and contrasted with different manipulations provoking cytokine deficiencies, namely the application of cytokine antagonists, neutralizing antibodies and receptor derivates, receptor-targeted cytotoxic drugs, and germ line disruption of cytokine genes. Both types of intervention are afflicted with major problems. Whereas providing an excess of cytokines in vivo, especially at the systemic level, constitutes an essentially non-physiological intervention, the induction of cytokine deficiencies will only unravel essential effects, but is incapable of elucidating the many pleiotropic cytokine effects that, by virtue of the redundancy of the system, compensate for each other.

In vivo administration of interleukin-2 turns on anergic self-reactive T cells and leads to autoimmune disease.

One major mechanism of self tolerance involves the deletion of T cell clones in the thymus. In athymic mice, tolerance to self antigens must be generated extrathymically. T cells with self-reactive receptors undergo either peripheral clonal deletion or become unresponsive (i.e. anergic). The unresponsive state of human and mouse T cell clones in vitro can be reversed by the addition of exogenous interleukin (IL)-2, thus transforming anergic T cells to an activated state. Here it is shown that the in vivo delivery of IL-2 to athymic BALB/c nu/nu mice abrogates the anergic state of self-reactive V beta 3+ and V beta 11+ T cells [which are normally deleted in the minor lymphocyte stimulatory (Mls)-1b-, I-E(+)-expressing euthymic counterparts]. Thus, V beta 3+ and V beta 11+ T cells from IL-2-treated nude mice proliferate in response to T cell receptor cross-linking and acquire effector functions as measured by their ability to deliver aid to B cells upon specific stimulation. This activation correlates with the development of autoimmune manifestations (DNA autoantibodies, rheumatoid factors, erythroleukopenia and minimal change nephritis) in these IL-2-treated mice.

Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway.

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB-reactive T cells bearing T cell receptor (TcR) V beta 8 gene products. Given that interleukin 2 (IL-2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL-2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL-2 modulate SEB responses in vivo. Surprisingly, neither IL-2 nor CsA were able to change the in vivo kinetics and magnitude of SEB-induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB-reactive TcR V beta 8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing "anergy" and apoptotic cell death of V beta 8+ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V beta 8+ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)alpha chains in vivo, although SEB induces IL-2R alpha in vitro. Altogether, these results indicate that the IL-2/IL-2R-mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of V beta 8+ T cells from SEB-primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL-2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.

Interleukin 2 abrogates the nonresponsive state of T cells expressing a forbidden T cell receptor repertoire and induces autoimmune disease in neonatally thymectomized mice.

Under physiological conditions, the vast majority of T cells differentiate in the thymus, an organ that provides an optimal microenvironment for T cell maturation and shapes the T cell repertoire via positive and negative selection processes. In the present report, we demonstrate that neonatal thymectomy of CBA/H mice results in a diminution of T cells in peripheral lymphoid organs (spleen, lymph nodes), but is followed by a marked transient (12 wk) increase in Thy-1+ CD3+ cells in the peritoneal cavity. These cells exhibit predominantly a double-negative (CD4-CD8-) phenotype among which products of the T cell receptor (TCR) V beta 11 gene family (i.e., an I-E-reactive TCR normally deleted in I-E-bearing CBA/H mice) are selectively overexpressed. This observation suggests that, under athymic conditions, T cell differentiation and/or accumulation may occur in the peritoneal cavity. Intraperitoneal inoculation of an interleukin 2 (IL-2) vaccinia virus construct that releases high titers of human IL-2 in vivo induces conversion of these double-negative T cells to either CD4+ CD8- or CD4- CD8+ single positives, and allows in vitro stimulation of TCR V beta 11-bearing cells with a clonotypic anti-V beta antibody. Since IL-2 induces autoimmune manifestations (DNA autoantibodies, rheumatoid factors, and interstitial nephritis) in thymectomized CBA/H mice, but not in sham-treated littermates, this lymphokine is likely to enhance the autoaggressive function of T cells that bear forbidden, potentially autoreactive TCR gene products and that are normally deleted in the thymus.