Megan Barnden

We describe the generation of ovalbumin (OVA)-specific, MHC class II-restricted alpha beta T cell receptor (TCR) transgenic mice. Initial attempts at generating these transgenic mice utilized heterologous regulatory elements to drive the expression of cDNA genes encoding the separate alpha- and beta-chains of the TCR. Unexpectedly, T cells bearing the transgenic alpha beta TCR failed to emerge from the thymus in these mice, although the transgenes did modify endogenous TCR expression. However, subsequent modification of the approach which enabled expression of the TCR beta-chain under the control of its natural regulatory elements generated mice whose peripheral T cells expressed the transgenic TCR and were capable of antigen-dependent proliferation. These results show that successful generation of MHC class II-restricted, OVA-specific alpha beta TCR transgenic mice was dependent upon combining cDNA- and genomic DNA-based constructs for expression of the respective alpha- and beta-chains of the TCR.

Self-antigens expressed in extrathymic tissues such as the pancreas can be transported to draining lymph nodes and presented in a class I-restricted manner by bone marrow-derived antigen-presenting cells. Such cross-presentation of self-antigens leads to CD8+ T cell tolerance induction via deletion. In this report, we investigate the influence of CD4+ T cell help on this process. Small numbers of autoreactive OVA-specific CD8+ T cells were unable to cause diabetes when adoptively transferred into mice expressing ovalbumin in the pancreatic beta cells. Coinjection of OVA-specific CD4+ helper T cells, however, led to diabetes in a large proportion of mice (68%), suggesting that provision of help favored induction of autoimmunity. Analysis of the fate of CD8+ T cells indicated that CD4(+) T cell help impaired their deletion. These data indicate that control of such help is critical for the maintenance of CD8+ T cell tolerance induced by cross-presentation.

The present report provides the first extensive characterization of the OT-I TCR transgenic line, which produces MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). These cells are shown to be positively selected in vivo in H-2b C57BL/6 mice and in bm5 mice, which express the Kbm5 mutant molecule. In contrast, OT-I cells were not selected by mutant Kb molecules in bm1, bm3, bm8, bm10, bm11 or bm23 mice. Interestingly, however, when positive selection was examined in vitro in foetal thymic organ culture (FTOC), bm1 and bm8 were still poorly selective, but the bm3 haplotype now selected as efficiently as B6. The ability to select in vitro correlated with the capacity to present the ovalbumin (OVA) peptide to OT-I cells, as measured by induction of an OVA-specific proliferative response. These results suggest that a lower affinity TCR:MHC interaction may be necessary for positive selection in FTOC compared with selection in situ.

Acute myeloid leukemia (AML) blasts express high levels of interlekin-3 (IL-3) receptor-α (CD123). CSL360 is a recombinant, chimeric immunoglobulin G1 (IgG1), anti-CD123 monoclonal antibody (MoAb) that neutralizes IL-3 and demonstrates anti-leukemic activity in vitro. This phase 1 study assessed safety, pharmacokinetics and bioactivity of weekly intravenous CSL360 for 12 weeks in 40 patients with advanced AML across five dose levels (0.1-10.0 mg/kg). Other than mild infusion reactions, CSL360 was well tolerated. The maximal tolerated dose was not reached. The half-life was 4.9 days, and the area under the curve (AUC) and maximum concentration (Cmax) increased proportionally with dose. Doses ≥ 3.0 mg/kg resulted in complete saturation and down-regulation of CD123 and abolition of ex vivo proliferative responsiveness to IL-3, indicating adequate blockade of IL-3 signaling. Two patients responded, with one remaining in complete remission after 17 doses. CSL360 bound CD123 specifically, but did not induce anti-leukemic activity in most patients. While safe, MoAb blockade of CD123 function is insufficient as a therapeutic strategy.