Aurelian

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University of Maryland Baltimore
Experimental Therapeutics Symposium 2009

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Aurelian

  1. 1. HSV INFECTION OF STEM CELLS MAY CAUSE GVHD IN TRANSPLANT RECIPIENTS <ul><li>Shvartsbeyn M, Akpek G and Aurelian L </li></ul><ul><li>Department of Pharmacology and Experimental Therapeutics and Greenebaum Cancer Center </li></ul>
  2. 2. Stem cell therapies are not without complications <ul><li>Develop appropriate therapeutic strategies </li></ul><ul><li>Graft vs host disease (GVHD) is a major cause of late morbidity and mortality in stem cell transplant (SCT) recipients </li></ul><ul><li>It shares clinical properties with HSV-associated erythema multiforme (HAEM) </li></ul>
  3. 3. HAEM <ul><li>Inflammatory skin lesion caused by HSV infection of CD34+ hematopoetic stem cells (SC). </li></ul><ul><li>Infection increases expression of E-cadherin (E-cad) which regulates SC numbers and stimulates their differentiation into antigen-presenting dendritic cells (DC) </li></ul><ul><li>SC fragment HSV DNA and rapidly clear it but they retain the Pol gene </li></ul><ul><li>Pol+ dendritic cells repopulate skin </li></ul><ul><li>Most subjects have HSV immune memory resulting in inflammatory lesions. </li></ul>
  4. 4. Stem cell differentiation into DC <ul><li>CD34+ stem cells differentiate into antigen presenting DC </li></ul><ul><li>These include CD1a+/CD14- and CD14+CD1a- cells </li></ul><ul><li>Langerhans cells (LC) are the resident DCs in skin </li></ul><ul><li>LC are CD1a+/E-cad+ members of the DC family. They originate from CD1a+/CD14- progenitors </li></ul><ul><li>CD14+/CD1a- progenitors give rise to a separate E-cad- DC type (non-LC DC). </li></ul><ul><li>CD34+ can also differentiate into CD11b+ LC. </li></ul>
  5. 5. Does HSV reactivated from latency by the immunosuppressive therapy that precedes SCT cause/contribute GVHD in SCT recipients through its ability to regulate the differentiation of CD34+ cells?
  6. 6. Study Design <ul><li>Thirteen recipients of allogeneic SCT </li></ul><ul><li>Had immunosuppressive therapy and oral acyclovir initiated on day 3 post-transplant (D+3) for HSV systemic/neurologic morbidity </li></ul><ul><li>Peripheral blood mononuclear cells (PBMC) and skin biopsies obtained at enrollment ( baseline) and during GVHD development </li></ul><ul><li>Double immunofluorescence and FACS analysis used to examine expression of Pol and E-cadherin in CD34+, CD14+ and CD1a+ cells </li></ul><ul><li>Thirteen recipients of allogeneic SCT were enrolled. They had immunosuppressive therapy and oral acyclovir initiated on day 3 after transplant (D+3). </li></ul><ul><li>Peripheral blood mononuclear cells (PBMC) and skin biopsies were obtained at the time of enrollment (baseline) and during lesion development. </li></ul><ul><li>Double immunofluorescence (IF) of skin tissues and flow cytometry (FACS) were used to examine expression of Pol, E-cadherin (associated with virus-induced differentiation), CD34, CD1a and CD14. </li></ul><ul><li>Thirteen recipients of allogeneic SCT were enrolled. They had immunosuppressive therapy and oral acyclovir initiated on day 3 after transplant (D+3). </li></ul><ul><li>Peripheral blood mononuclear cells (PBMC) and skin biopsies were obtained at the time of enrollment (baseline) and during lesion development. </li></ul><ul><li>Double immunofluorescence (IF) of skin tissues and flow cytometry (FACS) were used to examine expression of Pol, E-cadherin (associated with virus-induced differentiation), CD34, CD1a and CD14. </li></ul><ul><li>Thirteen recipients of allogeneic SCT were enrolled. They had immunosuppressive therapy and oral acyclovir initiated on day 3 after transplant (D+3). </li></ul><ul><li>Peripheral blood mononuclear cells (PBMC) and skin biopsies were obtained at the time of enrollment (baseline) and during lesion development. </li></ul><ul><li>Double immunofluorescence (IF) of skin tissues and flow cytometry (FACS) were used to examine expression of Pol, E-cadherin (associated with virus-induced differentiation), CD34, CD1a and CD14. </li></ul>
  7. 7. RESULTS <ul><li>Patients fell into 3 groups </li></ul><ul><li>Group I: 5 patients with skin rash histologically diagnosed as GVHD but positive for HSV Pol </li></ul><ul><li>Group II : 3 patients with skin rash that did not fit histopathologic criteria for GHVD ; one was positive for HSV Pol </li></ul><ul><li>Group III : 5 patients that did not develop cutaneous eruptions; one had elevated levels of circulating CD34+/Pol+ cells </li></ul>
  8. 8. <ul><li>Developed skin rash diagnosed as GVHD grade 1 on D+10, shortly after the onset of genital HSV-2 lesions, which did not respond to acyclovir treatment (D+5). </li></ul><ul><li>New rash diagnosed as GVHD grade 3 on D+33 </li></ul>Group I – patient 1
  9. 9. Rash diagnosed as GVHD grade 3 Group I – patient 1
  10. 10. <ul><li>Had circulating CD34+/Pol+ and CD34+/E-cad+ cells the % of which increased with time after initial lesion </li></ul><ul><li>Circulating CD14+/Pol+ cells peaked on D+33 at time of second lesion </li></ul><ul><li>D+33 lesional skin was Pol+ (42% Pol+ cells) and had Pol+ LC (CD11b+/Pol+ (80%) and CD1a+/Pol+ (66%)) </li></ul>Group I – patient 1
  11. 11. Group I – patient 1 Circulating cells
  12. 12. Baseline skin is Pol negative DERMIS EPI DERMIS
  13. 13. Lesional skin(D+33) has Pol+ keratinocytes, dermal cells and LC (80%)
  14. 14. Lesional skin(D+33) has Pol+ dermal, CD34+ and CD1a+ (LC) cells (66%)
  15. 15. <ul><li>D+19-22: Blanching erythematous papules coalescing into plaques with scattered pustules on upper chest and back and erythematous indurated scalp. H&E Dx: GVHD grade 2 </li></ul><ul><li>D+29: Rash spread to 70-80% body surface area (BSA); more confluent, less indurated, and very pruritic. </li></ul><ul><li>Over the next two weeks, the rash improved </li></ul><ul><li>D+ 51: new flare covering 15-20% BSA diagnosed by H&E as GVHD grade 3. </li></ul>Group I – patient 3
  16. 16. Group I – patient 3
  17. 17. <ul><li>Circulating CD1a+/Pol+ LC elevated when the rash flared. </li></ul><ul><li>Pol positivity increased with severity </li></ul><ul><li>Multiple CD1a+/Pol+ cells in the skin lesion when the rash worsened </li></ul>Group I – patient 3
  18. 18. Group I – patient 3 <ul><li>Infection/differentiation is early event </li></ul><ul><li>Pol+ cells circulate throughout disease </li></ul>Rash BSA CD34+/Pol+ CD34+/Ecad+ CD14+/Pol+ CD1a+/Pol+ D+3 None 3.2 2.5 41.3 ND D+19 (day 3 of rash) 30-40% 1.5 0.72 26.3 ND D+29 70-80% ND ND ND ND D+42 20-30% 0.52 0.24 1.2 ND D+51 30-40% 1.6 1.1 7.2 8.9 D+63 10-20% 0.04 0.13 7.1 0.14
  19. 19. Pol expression increases with lesion severity Pol expression in epidermis/dermis correlates with clinical, not GVHD severity Epidermis Dermis Day 3 0 0 Day 19 (30-40% BSA) GVHD grade 2 19.6 ± 4 9.1 ± 1.1 Day 29 (70-80% BSA) 63.6 ± 5.1 49.6 ± 4.6 Day 51 (20-30% BSA) GVHD grade 3 22.8 ± 2.6 11.3 ± 2.8
  20. 20. Pol is expressed in lesional skin Baseline Day 29 Epidermis Dermis
  21. 21. CD34+ cells and their differentiation products target Pol to lesional skin CD34+/Pol+ CD14+/Pol+ CD1a+/Pol+ (71%) Dermis
  22. 22. <ul><li>Rash Clinically similar to that in Group I patients </li></ul><ul><li>Does not have H&E features of GVHD </li></ul><ul><li>Pol + cells in epidermis (21.6%) </li></ul><ul><li>Rare CD34+/Pol+ cells in dermis </li></ul>Group II – patient 4
  23. 23. <ul><li>Skin lesions that follow allogeneic SCT can result from HSV infection of transplanted CD34+ cells which stimulates their differentiation into skin-homing DC through increased E-cadherin expression. </li></ul><ul><li>These DC carry HSV DNA fragments notably those encoding Pol antigen to the skin causing inflammatory lesions (skin rashes) </li></ul>CONCLUSION
  24. 24. <ul><li>HSV-associated skin rashes in SCT recipients are generally diagnosed as GVHD. </li></ul><ul><li>Skin rashes induced by HSV infection of CD34+ cells may also have GVHD- diagnosis </li></ul><ul><li>Pol expression in skin correlated with the severity of the rash, not GVHD grade. </li></ul><ul><li>HSV is not associated with GVHD development in the GI tract </li></ul>CONCLUSION
  25. 25. CONCLUSION <ul><li>These studies underscore potential clinical problems associated with the use of stem cells in human therapeutic strategies </li></ul><ul><li>Additional studies may help to modify existing clinical practices to include improved (parenteral) acyclovir treatment. </li></ul>The studies were supported by the Maryland Stem Cell Research Fund
  26. 26. Thank You

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