Mobilization of CD8+ Central Memory T-Cells with Enhanced Reconstitution Potential in Mice By a Combination of G-CSF and GMI-1271-Mediated E-Selectin Blockade

T-cells are critical mediators of immune defense against pathogens and cancer. Adoptive T cell immunotherapy and T-cell engineering have promising clinical applications but T cell survival and exhaustion are current limitations.   Central memory cells (T_CM  CD62L⁺ CCR7⁺) and their precursors, stem central memory T-cells (T_SCM) possess the stem-like properties needed to reconstitute and prolong an effective immune response long-term. These cells have been shown to significantly improve therapeutic efficacy of adoptive T-cell therapy.  The challenge remains to harvest good quality T_CM -cells for these immunotherapy approaches.

The bone marrow (BM) is the major reservoir of CD8⁺ T_CM  and their precursors. We have previously shown that E-selectin is expressed in the BM vasculature and drives activation and differentiation of hematopoietic stem cells during G-CSF induced mobilization to the blood. We find therapeutic blockade of E-selectin promotes HSC self-renewal and reconstitution in vivo. We now examine the impact of E-selectin blockade on CD8⁺ T cell mobilization from the bone marrow to the blood and hypothesize that E-selectin blockade may also dampen the activation/differentiation of this subset.

First we administered a standard G-CSF regime (filgastim 250ug/kg/day for 3 days) to mice and then dosed some cohorts with GMI-1271 (40mg/kg BID) from 12 to 72 hours within this 3 day period.  Administration of G-CSF alone results in a near complete disappearance of bone marrow resident CD8⁺ T_CM cells, and their apparent migration (increase in numbers) to the blood, while CD8⁺ subsets in the lymph nodes and spleen were barely affected by G-CSF. Furthermore among T-cell subsets, CD8⁺ but not CD4⁺ T_CM were specifically mobilized into the blood when GMI-1271 was co-administered for the last 12 to 24 hours of G-CSF. These findings are consistent with reports demonstrating the bone marrow to be a major reservoir for CD8⁺ but not CD4⁺ central memory T-cells.

Administration of GMI-1271 caused a marked enhancement in mobilization into the blood of CD8⁺ T_CM/SCM (CD62L^hi, CCR7⁺) cells over treatment with G-CSF alone (p<0.05).  To determine the functional consequences of this skewed mobilization following GMI-1271 co-administration, 25 uL of mobilized blood was transplanted into irradiated congenic B6.SJL recipients together with 2x10⁵ congenic BM cells to analyze long-term donor T-cell engraftment in the recipient mice. We found G-CSF mobilized donor blood did not contribute CD8⁺ T_CM cells that can persist post-transplant (<0.5% at 20 weeks post-transplant). In contrast when donor mice were mobilized with G-CSF together with E-selectin blockade (GMI-1271), we found elevated levels of donor blood derived CD8⁺ T-cells demonstrating robust long-term CD8⁺ T-cell persistence / regeneration (5.3 ±3.2% of total recipient T-cells, p=0.04). This dramatic boost in donor CD8⁺ T-cell reconstitution in mobilized blood following GMI-1271 co-administration is likely to be due to the long-term persistence and in vivo amplification of CD8⁺ T_CM cells from donor mobilized blood.  Similar in vivo enhancing effects of GMI-1271 were also observed with other mobilizing agents such as combined CXCR4 and VLA-4 blockade and GM-CSF resulting in a significant 4.9-fold boost in donor CD8⁺ reconstitution with GMI-1271. Importantly, only 12 hours of E-selectin blockade was sufficient to achieve this boost in CD8⁺ T_CM numbers in the blood following G-CSF.

In a previous report we have shown that therapeutic blockade of E-selectin promotes HSC self-renewal in vivo.  Thus, it is possible that E-selectin blockade boosts mobilization of CD8⁺ T_CM/SCM  with stem-like properties into the blood by loosening factors retaining CD8⁺ T_CM/SCM  in the bone marrow and/or blocking the E-selectin-mediated activation and differentiation of this T-cell subset.

In summary, our studies identify E-selectin blockade as a novel target to improve harvesting of CD8⁺ T_CM/SCM cells with stem-like properties. Blockade of this target with GMI-1271 significantly improves the in vivo reconstitution potential and regenerative properties of CD8⁺ T-cells from donor blood allowing a valuable source of desired T-cells for use in adoptive immunotherapy and T-cell engineering.