Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells.

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.

Membrane lymphotoxin is required for the development of different subpopulations of NK T cells.

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTalpha and LTbeta, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTalpha-/- and LTbeta-/- mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTalpha-/- and LTbeta-/- mice did not respond to the lipoglycan alpha-galactosylceramide, which is presented by mouse CD1 to Valpha14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and alpha-galactosylceramide reactive and those that are not, were affected by disruption of the LTalpha and LTbeta genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTbeta receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.

Targeted disruption of Traf5 gene causes defects in CD40- and CD27-mediated lymphocyte activation.

TRAF5 [tumor necrosis factor (TNF) receptor-associated factor 5] is implicated in NF-kappaB and c-Jun NH(2)-terminal kinase/stress-activated protein kinase activation by members of the TNF receptor superfamily, including CD27, CD30, CD40, and lymphotoxin-beta receptor. To investigate the functional role of TRAF5 in vivo, we generated TRAF5-deficient mice by gene targeting. Activation of either NF-kappaB or c-Jun NH(2)-terminal kinase/stress-activated protein kinase by tumor necrosis factor, CD27, and CD40 was not abrogated in traf5(-/-) mice. However, traf5(-/-) B cells showed defects in proliferation and up-regulation of various surface molecules, including CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. Moreover, in vitro Ig production of traf5(-/-) B cells stimulated with anti-CD40 plus IL-4 was reduced substantially. CD27-mediated costimulatory signal also was impaired in traf5(-/-) T cells. Collectively, these results demonstrate that TRAF5 is involved in CD40- and CD27-mediated signaling.

Human TNF receptor-associated factor 5 (TRAF5): cDNA cloning, expression and assignment of the TRAF5 gene to chromosome 1q32.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for members of the TNF receptor superfamily. We previously identified murine TRAF5 (mTRAF5) and showed that it specifically interacts with the lymphotoxin-beta receptor (LT-beta R) and activates the transcription factor NF-kappa B. Here we have cloned the human TRAF5 homologue (hTRAF5) by cross hybridization with mTRAF5 cDNA. hTRAF5 cDNA is composed of 2894 nucleotides with a 557-amino-acid open reading frame that exhibits 77.5 and 80% identity to mTRAF5 at the nucleotide and amino acid levels, respectively. Northern blot analysis revealed that hTRAF5 mRNA is expressed in all visceral organs. Western blotting revealed that hTRAF5 protein was abundantly expressed in the human follicular dentritic cell line, FDC-1, and to a much lesser degree in several tumor cell lines. Interspecific backcross mapping revealed that Traf5 is located in the distal region of mouse chromosome 1, which shares a region of homology with human chromosome 1q. Fluorescence in situ hybridization confirmed regional localization to human chromosome 1q32.

Human tumor necrosis factor receptor p75/80 (CD120b) gene structure and promoter characterization.

Tumor necrosis factor receptor p75 (TNF-R p75) is a 75-kDa type I transmembrane protein expressed predominantly on cells of hematopoietic lineage. TNF-R p75 belongs to the TNF receptor superfamily characterized by cysteine-rich extracellular regions composed of three to six disulfide-linked domains. In the present report we have characterized, for the first time, the complete gene structure for human TNF-R p75, which spans approximately 43 kbp. The gene consists of 10 exons (ranging from 34 base pairs to 2.5 kilobase pairs) and nine introns (343 base pairs to 19 kilobase pairs). Consensus elements for transcription factors involved in T cell development and activation were noted in the 5'-flanking region including T cell factor-1, Ikaros, AP-1, CK-2, interleukin-6 receptor E (IL-6RE), ISRE, GAS, NF-kappaB, and Sp1. The unusual (GATA)n and (GAA)(GGA) repeats found within intron 1 may prove useful for further genome analysis within the 1p36 chromosomal locus. Characterization of the human TNF-R p75 gene structure will permit further assessment of its involvement in normal hematopoietic cell development and function, autoimmune disease, and nonrandom translocations in hematopoietic malignancies.

Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression.

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

NG-methyl-L-arginine, an inhibitor of nitric oxide formation, reverses IL-2-mediated hypotension in dogs.

The effects of NG-methyl-L-arginine (L-NMA), an inhibitor of nitric oxide formation, were studied in dogs treated with interleukin-2 (IL-2). The administration of IL-2 to dogs resulted in hypotension within 3 days of treatment. The development of hypotension correlated with accumulation in the serum of nitrate, which is a stable breakdown product of nitric oxide. Administration of L-NMA decreased serum nitrate levels and increased the mean arterial pressure. The antihypotensive effect was dose dependent with a maximum effect observed at a dose of 20 mg/kg. Administration of a continuous infusion of L-NMA (5 mg.kg-1.h-1) maintained the mean arterial pressure for 48 h with concurrent administration of IL-2. Evaluation of IL-2-induced lymphokine-activated killer cell proliferation and tumoricidal activity toward a canine glioblastoma target cell line was unaffected by L-NMA. These studies imply that L-NMA may effectively ameliorate the dose-limiting hypotension associated with administration of IL-2 without adversely affecting the antitumor effects.

Role of tumor necrosis factor species in the sequence-dependent effects of interleukin-2--tumor necrosis factor immunotherapy in mice: implications for preclinical cytokine testing.

We previously demonstrated that recombinant human tumor necrosis factor (hTNF) is synergistic with human interleukin-2 (hIL-2) for in vivo regression of murine tumors. In mice, the timing of cytokine administration is critical in achieving synergy. Because hTNF exhibits negligible binding to the type II murine TNF receptor (TNF-R), we questioned whether murine TNF (mTNF) would have therapeutic benefits, scheduling requirements, and toxic effects similar to those of the hIL-2-hTNF combination. To evaluate the biological effects of TNF-R types I and II interaction, we directly compared the effects of mTNF and hTNF in combination with hIL-2 on in vivo tumor regression and in vitro activation of murine splenocytes. Our results demonstrate for the first time that (a) the cytokine combination hTNF-hIL-2 is consistently more efficacious than mTNF-hIL-2 in in vivo murine immunotherapy models; (b) the in vivo antitumor effects of hTNF-hIL-2 and not mTNF-hIL-2 are critically dependent upon cytokine scheduling; and (c) in vitro culture of splenocytes with mTNF-hIL-2 enhances cellular proliferation, Lyt 2, and TNF-RI expression compared with hTNF-hIL-2. Collectively, these studies extend the previous findings of species-specific mTNF-R responses and reveal that optimal scheduling and efficacy of TNF-hIL-2 combination therapy in murine tumor models is critically dependent upon the TNF species employed.