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Generation of functional CLL-specific cord blood CTL using CD40-ligated CLL APC.

Decker WK, Shah N, Xing D, Lapushin R, Li S, Robinson SN, Yang H, Parmar S, Halpert MM, Keating MJ, Gribben JG, Molldrem JJ, Shpall EJ, Wierda WG - PLoS ONE (2012)

Bottom Line: The generation of CLL-specific CTL from UCB T-cells, primed and expanded against the leukemic clone, might enhance the GVL effect and improve outcomes with UCB transplantation.Subsequently, we developed a clinically-applicable procedure permitting separation of alloreactive CTL from leukemia-specific CTL.Leukemia-specific CTL were able to mediate in vivo killing of CLL in humanized mice without concurrent or subsequent development of xenoGVHD.

View Article: PubMed Central - PubMed

Affiliation: Baylor College of Medicine, Department of Pathology and Immunology, Houston, Texas, United States of America. decker@bcm.edu

ABSTRACT
Though remissions have been observed following allo-HSCT for the treatment of CLL, many CLL patients are ineligible for transplant due to the lack of HLA-compatible donors. The use of umbilical cord blood (UCB) permits transplantation of many patients who lack HLA-compatible donors due to reduced requirements for stringent HLA matching between graft and recipient; however, disease relapse remains a concern with this modality. The generation of CLL-specific CTL from UCB T-cells, primed and expanded against the leukemic clone, might enhance the GVL effect and improve outcomes with UCB transplantation. Here we report the generation of functional, CLL-specific CTL using CD40-ligated CLL cells to prime partially-HLA matched UCB T-cells. Functionality and specificity were demonstrated by immune synapse assay, IFN-γ ELISpot, multi-parametric intracellular cytokine flow cytometry, and (51)Cr release assay. The use of patient-specific, non-CLL controls demonstrated the generation of both alloantigen and CLL-specific responses. Subsequently, we developed a clinically-applicable procedure permitting separation of alloreactive CTL from leukemia-specific CTL. Leukemia-specific CTL were able to mediate in vivo killing of CLL in humanized mice without concurrent or subsequent development of xenoGVHD. Our results demonstrate that generation of CLL-specific effectors from UCB is feasible and practical, and the results support further exploration of this strategy as a treatment modality for CLL.

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In contrast to UCB-derived T-cells, adult-derived T-cells do not demonstrate antigenic specificity against unligated CLL targets.A. IFN-γ ELISpot demonstrates no differences in immune reactivity between adult T-cells primed by CLL-APC and CD8+ targets or CLL targets. Y axis = IFN-γ ELISpots/50,000 cells. B. 51Cr killing assay shows adult T-cells primed by CLL-APC mediate higher levels of killing against the normal alloantigenic target (CD8+ cells) than against the unligated CLL target. Y axis = percent specific killing. For both panels, error bars = +/−SD.
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pone-0051390-g006: In contrast to UCB-derived T-cells, adult-derived T-cells do not demonstrate antigenic specificity against unligated CLL targets.A. IFN-γ ELISpot demonstrates no differences in immune reactivity between adult T-cells primed by CLL-APC and CD8+ targets or CLL targets. Y axis = IFN-γ ELISpots/50,000 cells. B. 51Cr killing assay shows adult T-cells primed by CLL-APC mediate higher levels of killing against the normal alloantigenic target (CD8+ cells) than against the unligated CLL target. Y axis = percent specific killing. For both panels, error bars = +/−SD.

Mentions: Previous reports have indicated defects in the ability of adult (i.e. non-neonatal) peripheral blood-derived T-cells to interact immunologically with CLL targets in the absence of previous CD40 ligation. In other words, though the strategy of CD154 transfection and subsequent CD40 ligation has previously been used to generate CLL-APC and CLL-specific T-cells, such T-cells have typically been able to react only against CD154-transduced CLL targets but not against native, unmanipulated CLL targets[17]; [20]; [21]; [23]. In order to determine whether the use of cord blood T-cells genuinely imparts a distinct immunological advantage over the use of adult peripheral blood-derived T-cells, experiments were repeated using adult peripheral blood apheresis products matched at 4/6 HLA-A,B, or DR loci with patient CLL cells. Execution of this experiment was technically very challenging as there were relatively few normal donor apheresis products available, hence 4/6 HLA-matching to CLL patient cells was difficult to achieve. Ultimately, two different normal donor apheresis products were matched at 4/6 HLA loci to a single CLL patient. One of these normal donor products failed to expand at all when co-cultured with CD40-ligated CLL cells, a phenomenon that was not observed among 14 different co-culture experiments using UCB-derived T-cells. The second normal donor product expanded and was able to be analyzed. IFN-γ ELISpot indicated that adult-derived PBMCs could recognize alloantigen; however, there was no indication that CLL antigens were recognized with any kind of specificity (Figure 6A). Further, adult PBMC-derived T-cells primed by CLL-APCs were also unable to mitigate significant lysis of CLL cells, indeed, demonstrating a preferential lysis of the autologous CD8+ cells rather than the CLL cells against which they had been primed (Figure 6B). This lytic pattern was not observed among any of the experiments performed with partially-HLA matched UCB T-cells.


Generation of functional CLL-specific cord blood CTL using CD40-ligated CLL APC.

Decker WK, Shah N, Xing D, Lapushin R, Li S, Robinson SN, Yang H, Parmar S, Halpert MM, Keating MJ, Gribben JG, Molldrem JJ, Shpall EJ, Wierda WG - PLoS ONE (2012)

In contrast to UCB-derived T-cells, adult-derived T-cells do not demonstrate antigenic specificity against unligated CLL targets.A. IFN-γ ELISpot demonstrates no differences in immune reactivity between adult T-cells primed by CLL-APC and CD8+ targets or CLL targets. Y axis = IFN-γ ELISpots/50,000 cells. B. 51Cr killing assay shows adult T-cells primed by CLL-APC mediate higher levels of killing against the normal alloantigenic target (CD8+ cells) than against the unligated CLL target. Y axis = percent specific killing. For both panels, error bars = +/−SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526610&req=5

pone-0051390-g006: In contrast to UCB-derived T-cells, adult-derived T-cells do not demonstrate antigenic specificity against unligated CLL targets.A. IFN-γ ELISpot demonstrates no differences in immune reactivity between adult T-cells primed by CLL-APC and CD8+ targets or CLL targets. Y axis = IFN-γ ELISpots/50,000 cells. B. 51Cr killing assay shows adult T-cells primed by CLL-APC mediate higher levels of killing against the normal alloantigenic target (CD8+ cells) than against the unligated CLL target. Y axis = percent specific killing. For both panels, error bars = +/−SD.
Mentions: Previous reports have indicated defects in the ability of adult (i.e. non-neonatal) peripheral blood-derived T-cells to interact immunologically with CLL targets in the absence of previous CD40 ligation. In other words, though the strategy of CD154 transfection and subsequent CD40 ligation has previously been used to generate CLL-APC and CLL-specific T-cells, such T-cells have typically been able to react only against CD154-transduced CLL targets but not against native, unmanipulated CLL targets[17]; [20]; [21]; [23]. In order to determine whether the use of cord blood T-cells genuinely imparts a distinct immunological advantage over the use of adult peripheral blood-derived T-cells, experiments were repeated using adult peripheral blood apheresis products matched at 4/6 HLA-A,B, or DR loci with patient CLL cells. Execution of this experiment was technically very challenging as there were relatively few normal donor apheresis products available, hence 4/6 HLA-matching to CLL patient cells was difficult to achieve. Ultimately, two different normal donor apheresis products were matched at 4/6 HLA loci to a single CLL patient. One of these normal donor products failed to expand at all when co-cultured with CD40-ligated CLL cells, a phenomenon that was not observed among 14 different co-culture experiments using UCB-derived T-cells. The second normal donor product expanded and was able to be analyzed. IFN-γ ELISpot indicated that adult-derived PBMCs could recognize alloantigen; however, there was no indication that CLL antigens were recognized with any kind of specificity (Figure 6A). Further, adult PBMC-derived T-cells primed by CLL-APCs were also unable to mitigate significant lysis of CLL cells, indeed, demonstrating a preferential lysis of the autologous CD8+ cells rather than the CLL cells against which they had been primed (Figure 6B). This lytic pattern was not observed among any of the experiments performed with partially-HLA matched UCB T-cells.

Bottom Line: The generation of CLL-specific CTL from UCB T-cells, primed and expanded against the leukemic clone, might enhance the GVL effect and improve outcomes with UCB transplantation.Subsequently, we developed a clinically-applicable procedure permitting separation of alloreactive CTL from leukemia-specific CTL.Leukemia-specific CTL were able to mediate in vivo killing of CLL in humanized mice without concurrent or subsequent development of xenoGVHD.

View Article: PubMed Central - PubMed

Affiliation: Baylor College of Medicine, Department of Pathology and Immunology, Houston, Texas, United States of America. decker@bcm.edu

ABSTRACT
Though remissions have been observed following allo-HSCT for the treatment of CLL, many CLL patients are ineligible for transplant due to the lack of HLA-compatible donors. The use of umbilical cord blood (UCB) permits transplantation of many patients who lack HLA-compatible donors due to reduced requirements for stringent HLA matching between graft and recipient; however, disease relapse remains a concern with this modality. The generation of CLL-specific CTL from UCB T-cells, primed and expanded against the leukemic clone, might enhance the GVL effect and improve outcomes with UCB transplantation. Here we report the generation of functional, CLL-specific CTL using CD40-ligated CLL cells to prime partially-HLA matched UCB T-cells. Functionality and specificity were demonstrated by immune synapse assay, IFN-γ ELISpot, multi-parametric intracellular cytokine flow cytometry, and (51)Cr release assay. The use of patient-specific, non-CLL controls demonstrated the generation of both alloantigen and CLL-specific responses. Subsequently, we developed a clinically-applicable procedure permitting separation of alloreactive CTL from leukemia-specific CTL. Leukemia-specific CTL were able to mediate in vivo killing of CLL in humanized mice without concurrent or subsequent development of xenoGVHD. Our results demonstrate that generation of CLL-specific effectors from UCB is feasible and practical, and the results support further exploration of this strategy as a treatment modality for CLL.

Show MeSH
Related in: MedlinePlus