Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope ± cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1α plus IL-18, IL-1α plus IL-12, and IL-1α plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60–71% peptide-specific 51Cr release). Peak H-2Dd-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8+ PBMC. Nasal immunization with HIV immunogen and IL-1α, IL-12, and GM-CSF also induced Ag-specific IFN-γ-secreting cells in the draining cervical lymph node and the lung. The use of IL-1α, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1α, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.

Genetic studies have demonstrated that the basic helix-loop-helix protein E2A is an essential transcription factor in B lymphocyte lineage commitment and differentiation. However, the mechanism underlying E2A-mediated transcription regulation is not fully understood. Here, we investigated the physical and genetic interactions between E2A and co-activators histone acetyltransferases (HATs) in B cells. Gel filtration analysis of human pre-B cell nuclear extract showed that E2A coelutes with the HATs p300, CBP, and PCAF. A co-immunoprecipitation assay further demonstrated that a fraction of endogenous E2A proteins is associated with each of the three HATs. We show that these HATs acetylate E2A in vitro, enhance E2A-mediated transcription activity, and promote nuclear retention of E2A proteins. A catalytic mutation of p300 completely abrogates the ability of p300 to acetylate E2A and to promote E2A nuclear retention in 293T cells. A breeding test between E2A heterozygous mice and p300 heterozygous mice demonstrated that these two genes interact for proper B cell development. Collectively, these results suggest that E2A and HATs collaboratively regulate B cell development.The development of B lymphocytes in the bone marrow is initiated and tightly regulated by at least three transcription factors, E2A, EBF, and Pax5 (1). Mice missing any one of these transcription factors show complete block in B cell development at the pro-B cell stage (2-5). Although the expression of EBF and Pax5 are relatively restricted to the B cell lineage, E2A is found to be much broadly expressed. Both biochemical and genetic analyses have indicated that E2A is the most upstream regulator among the three transcription factors and is continuously involved in regulating the expression of B cell-specific genes through the later stages of B cell development (6, 7). It is not clear how E2A controls the broad array of tissue-specific and stage-specific gene expression during B cell development.E2A is a founding member of the basic helix-loop-helix (bHLH) 1 transcription factor family, which plays an evolutionarily conserved role in regulating the differentiation events in various tissue types including B lymphocytes in mammals (8).The E2A gene encodes two bHLH transcription factors, E12 and E47, which are generated through differential splicing to two adjacent exons that encode the bHLH domains (9). The bHLH domains are required for protein dimerization and DNA binding (10). Two transactivation domains (AD) are mapped to the amino terminus of the E2A proteins (11, 12). E2A proteins form homodimers or heterodimers through HLH interactions with other broadly expressed bHLH transcription factors such as HEB (13) or with tissue-restricted bHLH transcription factors such as MyoD (14). These bHLH protein dimers bind to DNA at the consensus sequence CANNTG, designated as the E-box. Functional E-box sites are found in the promoters and enhancers of a wide variety of tissue-specific genes including immunoglobulin genes in B cells (15). The ubiquitously exp...

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Summary E2A has been shown to be an important transcription factor downstream of the T‐cell receptor (TCR) signal during T‐cell development. The TCR signal is known to elicit different cellular responses at different stages of T‐cell development. Whether E2A is still required for normal TCR signalling in mature T cells is unknown. Here we examined T‐cell function after disruption of the E2A gene exclusively in the T‐cell lineage. The conditional E2A‐deficient mice show enhanced humoral immunity to a T‐dependent antigen. We further show that E2A is involved in regulating TCR‐induced T‐cell proliferation events. However, E2A seems to play opposite roles in naïve and effector T cells. In the absence of E2A, TCR‐induced proliferation is increased in naïve T cells and decreased in effector T cells. At older ages, these mice frequently develop antinuclear antibodies and proteinuria. Our studies suggest that E2A regulates T‐cell function and the loss of E2A may promote age‐dependent autoimmune diseases.

Lymphocytes are derived from hematopoietic stem cells (HSC) following a series of regulated differentiation events. Multipotent HSCs become committed to the B cell lineage in bone marrow and the T cell lineage in the thymus after receiving appropriate signals from the corresponding microenvironment. These committed lymphoid cells must then undergo V(D)J recombination at the immunoglobulin gene or T cell receptor gene locus resulting in clonal production of functional B or T lymphocytes, respectively. Lymphocyte commitment and differentiation are accompanied by programmed gene expression or repression events which are driven by lineage and stage specific transcription factors. The basic-helix-loop-helix (bHLH) transcription factors encoded by the E2A gene are involved in several differentiation events during B and T cell development, including lineage commitment, initiation of V(D)J recombination, and antigen receptor mediated proliferation and differentiation. Several recent reviews have provided a comprehensive discussion of biochemical, cellular, and genetic research on E2A function in lymphocyte development (1,2). Here, we only discuss some of the genetic approaches our laboratory (except where it is noted) has undertaken to investigate the molecular pathways mediated by E2A transcription factors in lymphocyte development.