Michael K. Tanner

TNF-α Is Critical to Facilitate Hemopoietic Stem Cell Engraftment and Function

The use of tolerogenic cells as an approach to induce tolerance to solid organ allografts is being aggressively pursued. A major limitation to the clinical application of cell-based therapies has been the ability to obtain sufficient numbers and also preserve their tolerogenic state. We previously reported that small numbers of bone marrow-derived CD8+/TCR− graft facilitating cells (FC) significantly enhance hemopoietic stem cell (HSC) engraftment in allogeneic and syngeneic recipients. Although the majority of FC resemble precursor plasmacytoid dendritic cells (p-preDC), p-preDC do not replace FC in facilitating function. In the present studies, we investigated the mechanism of FC function. We show for the first time that FC significantly enhance HSC clonogenicity, increase the proportion of multipotent progenitors, and prevent apoptosis of HSC. These effects require direct cell:cell contact between FC and HSC. Separation of FC from HSC by transwell membranes completely abrogates the FC effect on HSC. p-preDC FC do not replace FC total in these effects on HSC function. FC produce TNF-α, and FC from TNF-α-deficient mice exhibit impaired facilitation in vivo and loss of the in vitro effects on HSC. Neutralizing TNF-α in FC similarly blocks the FC effect. The antiapoptotic effect of FC is associated with up-regulation of Bcl-3 transcripts in HSC and blocking of TNF-α is associated with abrogation of up-regulation of Bcl-3 transcripts. These data demonstrate a critical role for TNF-α in mediating FC function. FC may have a significant impact upon the safe use of chimerism to establish tolerance to transplanted organs and tissue.

CD8+, αβ-TCR+, and γδ-TCR+ Cells in the Recipient Hematopoietic Environment Mediate Resistance to Engraftment of Allogeneic Donor Bone Marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of αβ-TCR+, γδ-TCR+, αβ- plus γδ-TCR+, CD8+, or CD4+ cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4+ T cells. Mice lacking both αβ- and γδ-TCR+ cells engrafted without conditioning, suggesting that both αβ- and γδ-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8− cell was targeted by the CyP conditioning. The CD8+ cell effector function is mechanistically different from that for conventional T cells, and independent of CD4+ T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.

Flt3-ligand-mobilized peripheral blood, but not Flt3-ligand-expanded bone marrow, facilitating cells promote establishment of chimerism and tolerance.

Facilitating cells (CD8+/TCR−) (FCs) enhance engraftment of limiting numbers of hematopoietic stem cells (HSCs). The primary component of FCs is precursor‐plasmacytoid dendritic cells (p‐preDCs), a tolerogenic cell expanded by Flt3‐ligand (FL). In this study, we evaluated the function and composition of FL‐expanded FCs. FL treatment resulted in a significant increase of FCs in bone marrow (BM) and peripheral blood (PB). When FL‐expanded FCs were transplanted with c‐Kit+/Sca‐1+/Lin− (KSL) cells into allogeneic recipients, BM‐FCs exhibited significantly impaired function whereas PB‐FCs were potently functional. A significant upregulation of P‐selectin expression and downregulation of VCAM‐1 (vascular cell adhesion molecule 1) were present on FL‐expanded PB‐FCs compared with FL BM‐FCs. Stromal cell–derived factor‐1 (SDF‐1), and CXCR4 transcripts were significantly increased in FL PB‐FCs and decreased in FL BM‐FCs. Supernatant from FL PB‐FCs primed HSC migration to SDF‐1, confirming production of the protein product. The FL PB‐FCs contained a predominance of p‐preDCs and natural killer (NK)–FCs, and NK‐FCs were lacking in FL BM‐FCs. The impaired function for BM‐FCs was restored within 5 days after cessation of treatment. Taken together, these data suggest that FCs may enhance HSC homing and migration via the SDF‐1/CXCR4 axis and adhesion molecule modulation. These findings may have implications in development of strategies for retaining function of ex vivo manipulated FCs and HSCs.

Intranuclear staining of proteins in heterogeneous cell populations and verification of nuclear localization by flow cytometric analysis.

We describe techniques to detect nuclear transcription factors in thymocyte subsets using flow cytometry. We have adapted a method that minimizes autofluorescence of fixed cells, thereby allowing the detection of proteins expressed at low levels. An accompanying method in which the cytoplasm is removed from stained cells allows the confirmation of nuclear localization. These methods combine to provide a sensitive alternative approach for detecting nuclear proteins within heterogeneous cell populations.

Strategic Nonmyeloablative Conditioning: CD154:CD40 Costimulatory Blockade at Primary Bone Marrow Transplantation Promotes Engraftment for Secondary Bone Marrow Transplantation after Engraftment Failure

There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 costimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T cell depletion inhibits generation of the humoral immune response after primary BMT, as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, because almost all CFSE-labeled donor cells were killed at 0.5 and 3 h in sensitized recipients in in vivo cytotoxicity assay, reflecting Ab-mediated cytotoxicity. CD154:CD40 costimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.

Fms-Related Tyrosine Kinase 3 Expression Discriminates Hematopoietic Stem Cells Subpopulations With Differing Engraftment-Potential : Identifying the Most Potent Combination

Background. Fms-related tyrosine kinase 3 (Flt3)-ligand (FL) promotes the proliferation, differentiation, development, and mobilization of hematopoietic cells. We previously found that FL-mobilized hematopoietic stem cells (HSC) engraft efficiently, whereas FL-expanded bone marrow HSC do not. The function of FL-mobilized c-Kit+ Sca-1+Lin− (KSL) subpopulations has not been systematically evaluated. A precise definition of the repopulating ability is needed to define which HSC subpopulations are critical for long-term chimerism and tolerance induction. FL significantly mobilized c-Kithi and c-Kitlo Sca-1+Lin− cells into peripheral blood (PB). Here, we evaluated the influence of Flt3 expression on long-term repopulating ability of HSC subpopulations. Methods. c-Kithi or c-Kitlo KSL cells were sorted from PB of FL-treated green fluorescent protein-positive donors. The function of these cells was evaluated using competitive reconstitution assays, colony-forming units spleen, and colony forming cell assays. The function of c-Kithi CD34−Flt3− KSL, c-Kithi CD34+Flt3− KSL, c-Kithi CD34+Flt3+ KSL were investigated in an in vivo transplantation model. Results. Only FL-mobilized PB c-Kithi KSL cells exhibited high spleen colony-forming unit activity, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL-mobilization expanded both c-Kithi CD34+Flt3− cells (short-term HSC) and c-Kithi CD34−Flt3− KSL cells (long-term HSC). There was a significant decrease in c-Kithi CD34+Flt3+ KSL late multipotent progenitors in PB. A combination of c-Kithi CD34+Flt3− and c-Kithi CD34−Flt3− KSL cells offered the most effective rescue of ablated recipients. Conclusions. These data suggest that engraftment of purified HSC is influenced by both short- and long-term repopulating populations and that Flt3 expression may be useful for selecting the most critical HSC subpopulations for transplantation.