Immuno-PET of Murine T Cell Reconstitution Postadoptive Stem Cell Transplantation Using Anti-CD4 and Anti-CD8 Cys-Diabodies

Abstract

The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole body T lymphocyte dynamics non-invasively in vivo, we have generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for 89Zr-immunoPET detection of helper and cytotoxic T cell populations. Methods Anti-CD4 and -CD8 cys-diabodies were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cys-diabodies were site-specifically conjugated to maleimide-desferrioxamine for 89Zr radiolabeling and subsequent microPET/CT acquisition and ex vivo biodistribution in both wild type mice and a model of hematopoietic stem cell (HSC) transplantation. Results ImmunoPET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 weeks post-HSC transplant using the 89Zr-radiolabeled anti-CD4 and -CD8 cDbs. Conclusion These newly developed anti-CD4 and -CD8 immunoPET reagents represent a powerful resource to monitor T cell expansion, localization and novel engraftment protocols. Future potential applications of T cell targeted immunoPET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring.

ImmunoPET Imaging of Murine CD4+ T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging

“…We previously developed an anti-CD4 antibody fragment, GK1.5 cys-diabody (cDb), for immunoPET imaging of murine CD4 + T cells and described its use in monitoring CD4 + T cell reconstitution after hematopoietic stem cell transplantation [19]. GK1.5 cDb lacks the Fc region and clears rapidly though the kidney, enabling same- or next-day imaging.…”

Immuno-PET Imaging of Engineered Human T Cells in Tumors

“…In vivo imaging by Immuno-PET using antibody derivatives for targeting has been already clinically applied, although mainly for delineation of tumors (16). Recently, this technology has been also investigated to track murine T cells by targeting either general T-cell markers as CD4 and CD8 (17)(18)(19) or murine monoclonal TCR (20). However, these tools are mouse-specific and cannot be applied in humans.…”

Imaging T Cells In Vivo

“…Second, signal to noise in imaging procedures may decrease, if antibodies that target rare lymphocyte populations are used. On the other hand, the extensive knowledge available for the expression patterns of cluster of differentiation antigens on leukocytes in health and disease would deserve imaging investigations, with affinity reagents similar to the ones described in the current study (13). In this respect, CD4 and CD8 may represent only the beginning of a more extensive series of nuclear medicine investigations.…”

“…In this issue of The Journal of Nuclear Medicine, Anna Wu et al describe the successful imaging of secondary lymphoid organs, rich in T cells, using radiolabeled monoclonal antibodies, directed against the CD4 or CD8 T cell antigens (13). Specifically, the authors used antibodies in diabody format, which is cleared more rapidly than the corresponding parental IgG format and is thus more suitable for in vivo imaging applications (14,15).…”