Milken Institute School of Public Health Poster Presentations (Marvin Center & Video)

Title

Development of an Evaluation System for Immunological Tolerance In Sickle Cell Disease HSC Transplanted Patients with Stable Mixed Chimerism

Poster Number

69

Document Type

Poster

Status

Graduate Student - Masters

Abstract Category

Epidemiology and Biostatistics

Keywords

transplantation tolerance, sickle cell disease, lymphocyte, immunosuppression, immune reaction

Publication Date

Spring 2018

Abstract

Research in transplantation tolerance is robust; yet, tolerance induction remains elusive. Previously, we reported that the immunosuppressive drug sirolimus allowed long-term, stable mixed chimerism between donor and recipient cells in adult sickle cell disease (SCD) patients who underwent matched-sibling hematopoietic transplantation. Moreover, this state was maintained without graft rejection or graft-versus-host-disease even after withdrawal of the drug [NEJM 2007]. Here, we developed a novel assay using lymphocyte proliferation to evaluate tolerance in this unique patient population.

Methods:

Since we cannot separate donor cells from recipient cells in mixed chimeric patients, we developed a two-way mixed-lymphocyte reaction (MLR) using normal healthy donors to determine if proliferation could be measured when both cell populations are present within the same sample. As patients experience varying levels of donor chimerism, the proportion of one healthy donor to another spanned a full range of 1:9 to 9:1.

Next, to ensure that sirolimus would not impede in vitro proliferation, we challenged lymphocytes with sirolimus and measured proliferation over a 12 day course.

Finally, lymphocytes from 18 transplanted patients at various time points post-transplantation were split into two groups: on sirolimus - those who required continued immunosuppression per protocol - and off sirolimus - those who displayed tolerance - and evaluated proliferation.

Results:

Using the two-way MLR, lymphocyte proliferation from both healthy donors was able to be measured at each proportion tested, suggesting patient chimerism could be evaluated in both directions.

Sirolimus strongly inhibited lymphocyte proliferation in vitro during the first 6 days, but did not inhibit proliferation after 12 days, validating applicability in patients on sirolimus therapy.

Finally, patients on sirolimus were found to have on average 3-fold higher proliferation than an unstimulated control (p < 0.01) and 1.8-fold higher proliferation than patients off sirolimus (p < 0.05), while proliferation in patients off sirolimus did not differ from an unstimulated control (n.s.), reflecting tolerance. Patients with high levels of proliferation were predominately within the first year post-transplantation and had donor lymphocyte chimerism lower than 50%.

Conclusion:

Using this assay, lymphocyte proliferation in transplanted SCD patients was found to decrease to near normal levels after one year post-transplantation, suggesting tolerance occurs early during recovery. Further study of lymphocyte activity during this first year may reveal mechanisms leading to tolerance and enable patients to be weaned off of chronic immunosuppression without risk of rejection.

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Development of an Evaluation System for Immunological Tolerance In Sickle Cell Disease HSC Transplanted Patients with Stable Mixed Chimerism

Research in transplantation tolerance is robust; yet, tolerance induction remains elusive. Previously, we reported that the immunosuppressive drug sirolimus allowed long-term, stable mixed chimerism between donor and recipient cells in adult sickle cell disease (SCD) patients who underwent matched-sibling hematopoietic transplantation. Moreover, this state was maintained without graft rejection or graft-versus-host-disease even after withdrawal of the drug [NEJM 2007]. Here, we developed a novel assay using lymphocyte proliferation to evaluate tolerance in this unique patient population.

Methods:

Since we cannot separate donor cells from recipient cells in mixed chimeric patients, we developed a two-way mixed-lymphocyte reaction (MLR) using normal healthy donors to determine if proliferation could be measured when both cell populations are present within the same sample. As patients experience varying levels of donor chimerism, the proportion of one healthy donor to another spanned a full range of 1:9 to 9:1.

Next, to ensure that sirolimus would not impede in vitro proliferation, we challenged lymphocytes with sirolimus and measured proliferation over a 12 day course.

Finally, lymphocytes from 18 transplanted patients at various time points post-transplantation were split into two groups: on sirolimus - those who required continued immunosuppression per protocol - and off sirolimus - those who displayed tolerance - and evaluated proliferation.

Results:

Using the two-way MLR, lymphocyte proliferation from both healthy donors was able to be measured at each proportion tested, suggesting patient chimerism could be evaluated in both directions.

Sirolimus strongly inhibited lymphocyte proliferation in vitro during the first 6 days, but did not inhibit proliferation after 12 days, validating applicability in patients on sirolimus therapy.

Finally, patients on sirolimus were found to have on average 3-fold higher proliferation than an unstimulated control (p < 0.01) and 1.8-fold higher proliferation than patients off sirolimus (p < 0.05), while proliferation in patients off sirolimus did not differ from an unstimulated control (n.s.), reflecting tolerance. Patients with high levels of proliferation were predominately within the first year post-transplantation and had donor lymphocyte chimerism lower than 50%.

Conclusion:

Using this assay, lymphocyte proliferation in transplanted SCD patients was found to decrease to near normal levels after one year post-transplantation, suggesting tolerance occurs early during recovery. Further study of lymphocyte activity during this first year may reveal mechanisms leading to tolerance and enable patients to be weaned off of chronic immunosuppression without risk of rejection.