Susanne Luther

Tetramer monitoring to assess risk factors for recurrent cytomegalovirus reactivation and reconstitution of antiviral immunity post allogeneic hematopoietic stem cell transplantation

S. Borchers, S. Luther, U. Lips, N. Hahn, J. Kontsendorn, M. Stadler, S. Buchholz, H. Diedrich, M. Eder, U. Koehl, A. Ganser, E. Mischak‐ Weissinger. Tetramer monitoring to assess risk factors for recurrent cytomegalovirus reactivation and reconstitution of antiviral immunity post allogeneic hematopoietic stem cell transplantation.
Transpl Infect Dis 2011: 13: 222–236. All rights reserved

Expansion of recipient‐derived antiviral T cells may influence donor chimerism after allogeneic stem cell transplantation

Donor chimerism (DC) analysis is an important marker in the hematopoietic stem cell transplant follow‐up. Here, we present evidence for a possible relationship of infectious complications and declines in DC. We analyzed the DC in patients experiencing cytomegalovirus (CMV) reactivation. In addition, in some patients chimerism analyses of T‐cell subsets were performed. CMV‐specific cytotoxic T‐lymphocytes (CMV‐CTL) were monitored using human leukocyte antigen‐restricted multimer staining. Interestingly, CMV reactivation was accompanied by changes in DC in 11 of 67 patients transplanted. For example, DC declined in a cord blood recipient, in both total leukocytes and CD4 and CD8 T‐cell subsets upon CMV reactivation. The latter was controlled after only 5 days through expanding CMV‐CTL of 96% recipient origin, according to chimerism analysis of CMV‐CTL (enriched beyond 50%). In another patient, transplanted after reduced‐intensity conditioning from a DQB1 mismatched, CMV seronegative donor, incipient CMV reactivation was completely aborted by CMV‐CTL of recipient origin. However, at the same time, mixed chimerism dropped from 51% to 0% donor type, resulting in late graft rejection. Our data indicate that chimerism analyses in subset populations lead to a better understanding of declining total leukocyte chimerism. Furthermore, recipient‐derived CMV‐CTL may be able to control CMV reactivation after reduced‐intensity conditioning. We speculate that autologous CMV‐CTL may be instrumental to overcome recurrent CMV reactivations, especially in patients transplanted from CMV‐seronegative donors. In addition, the expansion of recipient‐derived CMV‐CTL may contribute to both, graft failure or to conversion to full DC.

Long term follow up of patients after allogeneic stem cell transplantation and transfusion of HSV-TK transduced T-cells.

Allogeneic stem cell transplantation (allo-HSCT) is one of the curative treatments for hematologic malignancies, but is hampered by severe complications, such as acute or chronic graft-versus-host-disease (aGvHD; cGvHD) and infections. CD34-selection of stem cells reduces the risk of aGvHD, but also leads to increased infectious complications and relapse. Thus, we studied the safety, efficacy, and feasibility of transfer of gene modified donor T-cells shortly after allo-HSCT in two clinical trials between 2002 and 2007 and here we compare the results to unmodified donor leukocyte infusion (DLI). The aim of these trials was to provide patients with the protection of T-cells after T-cell-depleted allo-HSCT in the matched or mismatched donor setting with an option to delete transduced T-cells, if severe aGvHD occurred within the trial period. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3, expressing HSV-TK and the truncated ΔLNGFR for selection of transduced cells. Transduced cells were transfused either after day +60 (matched donors) or on day +42 (haploidentical donors). Nine patients were included in the first trial (MHH; 2002 until 2007), two were included in TK007 (2005–2009) and six serves as a control group for outcome after haploidentical transplantation without HSV-TK-transduced DLI. Three patients developed acute GvHD, two had grade I of the skin, one had aGvHD on day +131 (post-HSCT; +89 post-HSV-TK DLI) grade II, which was successfully controlled by ganciclovir (GCV). Donor chimerism was stabilized after transfusion of the transduced cells in all patients treated. Functionality of HSV-TK gene expressing T-cells was shown by loss of bcr-able gene expression as well as by control of cytomegalovirus-reactivation. To date, six patients have relapsed and died, two after a second hematopoietic stem cell transplantation without T-cell depletion or administration of unmodified T-cells. Eleven patients (seven post-HSV-TK DLI) are alive and well to date.

Functional Analysis of a Single Chain Chimeric α/β-Granulocyte-Macrophage Colony-stimulating Factor Receptor IMPORTANCE OF A GLUTAMATE RESIDUE IN THE TRANSMEMBRANE REGION

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the α-subunit (α-GMR) to the intracellular part of the β-subunit (β-GMR) introducing an additional glutamate residue at the fusion site (α/β-GMR). We demonstrated the capacity of α/β-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this α/β-GMR is sufficient to mediate morphological changes, expression of α4- and β1-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of α/β-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when β-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not α/β-GMR can induce proliferation in the presence of an anti-α-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of α/β-GMR for ligand-induced receptor activation.

Possible Role of Minor H Antigens in the Persistence of Donor Chimerism after Stem Cell Transplantation; Relevance for Sustained Leukemia Remission

Persistent complete donor chimerism is an important clinical indicator for remissions of hematological malignancies after HLA-matched allogeneic stem cell transplantation (SCT). However, the mechanisms mediating the persistence of complete donor chimerism are poorly understood. The frequent coincidence of complete donor chimerism with graft-versus-leukemia effects and graft-versus-host disease suggests that immune responses against minor histocompatibility antigens (mHags) are playing an important role in suppressing the host hematopoiesis after allogeneic SCT. Here, we investigated a possible relationship between donor immune responses against the hematopoiesis-restricted mHag HA-1 and the long-term kinetics of host hematopoietic chimerism in a cohort of 10 patients after allogeneic HLA-matched, HA-1 mismatched SCT. Functional HA-1 specific CTLs (HA-1 CTLs) were detectable in 6/10 patients lysing host-type hematopoietic cells in vitro. Presence of HA-1 CTLs in the peripheral blood coincided with low host hematopoiesis levels quantified by highly sensitive mHag specific PCR. Additionally, co-incubation of host type CD34+ cells with HA-1 CTLs isolated after allogeneic SCT prevented progenitor and cobblestone area forming cell growth in vitro and human hematopoietic engraftment in immunodeficient mice. Conversely, absence or loss of HA-1 CTLs mostly coincided with high host hematopoiesis levels and/or relapse. In summary, in this first study, presence of HA-1 CTLs paralleled low host hematopoiesis levels. This coincidence might be supported by the capacity of HA-1 CTLs isolated after allogeneic SCT to specifically eliminate host type hematopoietic stem/progenitor cells. Additional studies involving multiple mismatched mHags in more patients are required to confirm this novel characteristic of mHag CTLs as factor for the persistence of complete donor chimerism and leukemia remission after allogeneic SCT.

Human CD8+ CD57- TEMRA cells: Too young to be called "old"

End-stage differentiation of antigen-specific T-cells may precede loss of immune responses against e.g. viral infections after allogeneic stem cell transplantation (SCT). Antigen-specific CD8+ T-cells detected by HLA/peptide multimers largely comprise CD45RA-/CCR7- effector memory (TEM) and CD45RA+/CCR7- TEMRA subsets. A majority of terminally differentiated T-cells is considered to be part of the heterogeneous TEMRA subset. The senescence marker CD57 has been functionally described in memory T-cells mainly composed of central memory (TCM) and TEM cells. However, its role specifically in TEMRA cells remained undefined. Here, we investigated the relevance of CD57 to separate human CD8+ TEMRA cells into functionally distinct subsets. CD57- CD8+ TEMRA cells isolated from healthy donors had considerably longer telomeres and showed significantly more BrdU uptake and IFN-γ release upon stimulation compared to the CD57+ counterpart. Cytomegalovirus (CMV) specific T-cells isolated from patients after allogeneic SCT were purified into CD57+ and CD57- TEMRA subsets. CMV specific CD57- TEMRA cells had longer telomeres and a considerably higher CMV peptide sensitivity in BrdU uptake and IFN-γ release assays compared to CD57+ TEMRA cells. In contrast, CD57+ and CD57- TEMRA cells showed comparable peptide specific cytotoxicity. Finally, CD57- CD8+ TEMRA cells partially changed phenotypically into TEM cells and gained CD57 expression, while CD57+ CD8+ TEMRA cells hardly changed phenotypically and showed considerable cell death after in vitro stimulation. To the best of our knowledge, these data show for the first time that CD57 separates CD8+ TEMRA cells into a terminally differentiated CD57+ population and a so far functionally undescribed “young” CD57- TEMRA subset with high proliferative capacity and differentiation plasticity.

miR-625-3p is upregulated in CD8+ T cells during early immune reconstitution after allogeneic stem cell transplantation

Alloreactive CD8+ T-cells mediate the curative graft-versus-leukaemia effect, the anti-viral immunity and graft-versus-host-disease (GvHD) after allogeneic stem cell transplantation (SCT). Thus, immune reconstitution with CD8+ T-cells is critical for the outcome of patients after allogeneic SCT. Certain miRNAs such as miR-146a or miR-155 play an important role in the regulation of post-transplant immunity in mice. While some miRNAs e.g. miR-423 or miR-155 are regulated in plasma or full blood during acute GvHD also in man, the relevance and expression profile of miRNAs in T-cells after allogeneic SCT is unknown. miR-625-3p has recently been described to be overexpressed in colorectal malignancies where it promotes migration, invasion and apoptosis resistance. Since similar regulative functions in cancer and T-cells have been described for an increasing number of miRNAs, we assumed a role for the cancer-related miR-625-3p also in T-cells. Here, we studied miR-625-3p expression selectively in CD8+ T-cells both in vitro and during immune reconstitution after allogeneic SCT in man. T-cell receptor stimulation lead to miR-625-3p upregulation in human CD8+ T-cells in vitro. Maintenance of elevated miR-625-3p expression levels was dependent on ongoing T-cell proliferation and was abrogated by withdrawal of interleukin 2 or the mTOR inhibitor rapamycin. Finally, miR-625-3p expression was analyzed in human CD8+ T-cells purified from 137 peripheral blood samples longitudinally collected from 74 patients after allogeneic SCT. miR-625-3p expression was upregulated on day 25 and on day 45, i.e. during the early phase of CD8+ T-cell reconstitution after allogeneic SCT and subsequently declined with completion of CD8+ T-cell reconstitution until day 150. In conclusion, this study has shown for the first time that miR-625-3p is regulated in CD8+ T-cells during proliferation in vitro and during early immune reconstitution after allogeneic SCT in vivo. These results warrant further studies to identify the targets and function of miR-625-3p in CD8+ T-cells and to analyze its predictive value for an effective immune reconstitution.

Characterization of High-Avidity Cytomegalovirus-Specific T Cells with Differential Tetramer Binding Coappearing after Allogeneic Stem Cell Transplantation

CMV reactivation is a major complication after allogeneic stem cell transplantation (SCT). Immune reconstitution of CMV-specific CTLs (CMV-CTLs) is essential for virus control. During CMV-CTL monitoring using mutated HLA/CMV tetramers selectively detecting high-avidity T cells, we observed coappearance of CMV-CTLs with low (CMV tetlow CTLs) and high tetramer binding (CMV tethigh CTLs) in 53/115 CMV IgG+ patients stem cell transplanted from CMV IgG+ donors. However, the relevance of these coappearing differentially tetramer binding (“dual”) CMV-CTLs was unclear. In this study, we investigated the kinetics, properties, and clinical impact of coappearing CMV tetlow and tethigh CTLs after allogeneic SCT. Patients with dual CMV-CTLs had more CMV tethigh than tetlow CTLs. Chimerism analysis of isolated CMV tetlow and tethigh CTLs revealed their exclusive donor origin. CMV tetlow and tethigh CTLs had an identical effector memory CD45RA−CCR7− and CD45RA+CCR7− T cell distribution, equal differentiation, senescence, and exhaustion marker expression and were negative for regulatory CD8+ T cell markers. Isolated CMV tetlow and tethigh CTLs were equally sensitive to CMV peptides in IFN-γ release and cytotoxicity assays. However, CMV tethigh CTLs proliferated more in response to low CMV peptide concentrations than tetlow CTLs. TCR repertoire analysis revealed that CMV tetlow and tethigh CTLs use different TCRs. Finally, dual CMV-CTLs were not associated with CMV antigenemia. In conclusion, these data show for the first time, to our knowledge, that both CMV tetlow and tethigh CTLs are functional effector T cells differing by proliferation, numbers in peripheral blood, and probably by their precursors without increasing the CMV reactivation risk after allogeneic SCT.

Possible Impact of Cytomegalovirus-Specific CD8+ T Cells on Immune Reconstitution and Conversion to Complete Donor Chimerism after Allogeneic Stem Cell Transplantation

Complete donor chimerism is strongly associated with complete remission after allogeneic stem cell transplantation (allo-SCT) in patients with hematologic malignancies. Donor-derived allo-immune responses eliminate the residual host hematopoiesis and thereby mediate the conversion to complete donor chimerism. Recently, cytomegalovirus (CMV) reactivation was described to enhance overall T cell reconstitution, to increase graft-versus-host disease incidence, and to reduce the leukemia relapse risk. However, the link between CMV and allo-immune responses is still unclear. Here, we studied the relationship between CMV-specific immunity, overall T cell reconstitution, and residual host chimerism in 106 CMV-seropositive patients transplanted after reduced-intensity conditioning including antithymocyte globulin. In accordance with previous reports, the recovery of CMV-specific cytotoxic T cells (CMV-CTLs) was more frequent in CMV-seropositive recipients (R) transplanted from CMV-seropositive than from seronegative donors (D). However, once CMV-CTLs were detectable, the reconstitution of CMV-specific CTLs was comparable in CMV R+/D- and R+/D+ patients. CD3+ and CD8+ T cell reconstitution was significantly faster in patients with CMV-CTLs than in patients without CMV-CTLs both in the CMV R+/D- and R+/D+ setting. Moreover, CMV-CTL numbers correlated with CD3+ and CD8+ T cell numbers in both settings. Finally, presence of CMV-CTLs was associated with low host chimerism levels 3 months after allo-SCT. In conclusion, our data provide a first indication that CMV-CTLs in CMV-seropositive patients might trigger the reconstitution of T cells and allo-immune responses reflected by the conversion to complete donor chimerism.