Roman Ivanov

Identification of a 40S Ribosomal Protein S4–Derived H-Y Epitope Able to Elicit a Lymphoblast-Specific Cytotoxic T Lymphocyte Response

Purpose: The superior graft-versus-leukemia (GVL) effect of the female-to-male stem cell transplantation is partially independent from the concomitant graft-versus-host reactivity. However, the antigenic basis of this selective GVL response remains enigmatic, because no H-Y antigens with hematopoietic-restricted expression were identified. In this study, we report a novel H-Y epitope that is preferentially recognized on activated proliferating lymphocytes. Experimental Design: We generated a CTL clone YKIII.8 that showed reactivity toward male B*5201+ CD40-activated B cells, EBV-lymphoblastoid cell lines, and phytohemagglutinin-activated T-cell blasts but little or no reactivity toward fibroblasts, CD14+ cells, or unstimulated B and T cells. The antigen recognized by YKIII.8 was identified by screening of a cDNA expression library, and its pattern of expression was investigated. Results: cDNA of the male isoform of 40S ribosomal protein S4 was found to encode the antigenic peptide TIRYPDPVI, which was recognized by YKIII.8. Western blot analysis showed that rapidly proliferating cells overexpress the RPS4 protein in comparison with nonrecognized cell subsets. Retroviral transfer of YKIII.8 T-cell receptor resulted in preservation of the lymphoblast-specific reactivity pattern. Conclusion: Our findings suggest that CTL specific to certain epitopes of ubiquitously expressed H-Y antigens may specifically target lymphoblasts, contributing to the selective GVL effect of female-to-male stem cell transplantation.

Rebuilding Human Leukocyte Antigen Class II–Restricted Minor Histocompatibility Antigen Specificity in Recall Antigen-Specific T Cells by Adoptive T Cell Receptor Transfer: Implications for Adoptive Immunotherapy

Purpose: Donor T cells directed to hematopoietic minor histocompatibility antigens (mHag) are appealing tools for adoptive immunotherapy of hematological malignancies after allogeneic stem cell transplantation (allo-SCT). Toward the development of a convenient strategy for ex vivo generation of human leukocyte antigen (HLA) class II–restricted mHag-specific T cells, we evaluated the feasibility of rebuilding mHag-specific T cell functions in donor-derived recall antigen-specific T cells via T cell receptor (TCR) transfer. Experimental Design: TCR α- and β-chains of an HLA-DPB1*0401–restricted T-cell clone recognizing a multiple myeloma-associated mHag were retrovirally transferred into a tetanus toxoid (TT)–specific clone derived from the original stem cell donor. TCR double-transduced cells were compared with the parent mHag- and TT-specific clones for antigen specificity, cytokine secretion, and cytotoxic activity and were analyzed for their in vitro expansion capacity in a TT- or mHag-specific fashion. Results: mHag-TCR–transduced TT-specific cells displayed both TT and mHag specificity. Similar to the parent cells, they secreted Th-1 cytokines and exerted significant cytotoxic activity against TT-pulsed or mHag+ target cells, including multiple myeloma cells. A 4-week expansion of TCR-transduced cells via the TT-specific TCR had no negative influence on the mHag-specific cytotoxic activity and resulted in 10- to 100-fold better cell yields as compared with mHag-specific expansion. Conclusions: HLA class II–restricted, mHag-specific effector functions can be successfully reconstructed in donor-derived TT-specific T cells via TCR transfer. Effective expansion of these T cells via TT-specific TCRs illustrate the suitability of this strategy for ex vivo expansion and possibly for in vivo TT-specific reboosting of HLA class II–restricted immunotherapeutic T cells.

B-cell expansion in the presence of the novel 293-CD40L-sCD40L cell line allows the generation of large numbers of efficient xenoantigen-free APC

BACKGROUND CD40-activated B lymphocytes have been used successfully as potent APC for the induction of T-cell responses. However, the 3T3-CD40L cell line, regularly used for engagement of CD40 on the B-cell surface, is a potential source of xenoantigens. This may affect the specificity of T cells stimulated with CD40-activated B cells, especially when generation of T-cell lines specific for endogenously processed Ag is desired. METHODS To develop a system that allows efficient expansion of B cells in the absence of sources of xenoantigens, we created a human 293-CD40L-sCD40L cell line that produces soluble CD40L and expresses CD40L on the cell surface. B cells from patients with hematologic malignancies were expanded on the 293-CD40L-sCD40L cells and used for stimulation of either naive or in vivo primed donor T cells in three HLA-identical patient-donor combinations. RESULTS The 293-CD40L-sCD40L cell line was able to stimulate B-cell growth with an efficiency superior to that of the commonly used 3T3-CD40L cell line. In all cases T-cell lines and, subsequently, T-cell clones were generated that showed reactivity against patient and not donor B cells, suggesting their specificity for minor histocompatibility antigens (mHAg). DISCUSSION B cells activated with GMP grade 293-CD40L-sCD40L can be used in a variety of applications. In particular, they may be suitable for ex vivo stimulation of T cells prior to donor lymphocyte infusion (DLI), which may enhance its graft versus leukemia (GvL) effect.

UTY‐specific TCR‐transfer generates potential graft‐versus‐leukaemia effector T cells

Immunotherapeutic approaches that target antigens that are differentially recognized on haematopoietic and non‐haematopoietic cells may specifically enhance the graft‐versus‐leukaemia (GVL) effect of donor lymphocyte infusion. In this study, we have characterized a new HLA‐B*5201‐restricted epitope of the UTY gene. Unusually, presentation of this epitope was restricted to lymphoblasts. As a result, a T cell clone specific to this epitope recognized normal and malignant male B and T lymphoblasts, while showing little reactivity towards male HLA‐B*5201+ fibroblasts. Transfer of its T cell receptor (TCR) into donor T cells led to the generation of large numbers of T cells, which acquired the specificity of the original clone, its avidity and the differential pattern of reactivity towards lymphoblasts and fibroblasts. Remarkably, the specific response of TCR‐transferred T cells was significantly higher than that of the original clone. This is the first demonstration of the possibility to preserve the specific pattern of a T cell response to a differentially expressed antigen after TCR‐transfer and to augment the amplitude of this response concomitantly. These results indicate that it may be feasible to enhance the GVL effect of donor lymphocyte infusions in lymphoproliferative malignancies by the transfer of TCRs specific to epitopes that are differentially recognized on lymphoblasts.

T cell receptor-transgenic primary T cells as a tool for discovery of leukaemia-associated antigens

Identification of a broad array of leukaemia‐associated antigens is a crucial step towards immunotherapy of haematological malignancies. However, it is frequently hampered by the decrease of proliferative potential and functional activity of T cell clones used for screening procedures. Transfer of the genes encoding the T cell receptor (TCR) α and β chains of leukaemia‐specific clones into primary T cells may help to circumvent this obstacle. In this study, transfer of two minor histocompatibility antigen (minor H antigen)‐specific TCRs was performed and the feasibility of the use of TCR‐transgenic T cells for identification of minor H antigens through cDNA library screening was investigated. We found that TCR‐transgenic cells acquired the specificity of the original clones and matched their sensitivity. Moreover, the higher scale of cytokine‐production by TCR‐transgenic T cells permits the detection of either small amounts of antigen‐positive cells or cells expressing low amounts of an antigen. When applied in equal numbers, TCR‐transgenic T cells and the original T cell clones produced similar results in the screening of a cDNA library. However, the use of increased numbers of TCR‐transgenic T cells allowed detection of minute amounts of antigen, barely discernible by the T cell clone. In conclusion, TCR‐transfer generates a large amount of functional antigen‐specific cells suitable for screening of cDNA expression libraries for identification of cognate antigens.

Clinical and quality of life responses to high-dose chemotherapy plus autologous stem cell transplantation in patients with multiple sclerosis: two case reports

During the last several years high-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) has been established as a therapeutic option for multiple sclerosis (MS) patients. We report on the long-term effects of HDCT + ASCT in two female patients affected by secondary progressive and relapsing-remitting types of MS, respectively. As a result, disease stabilization was achieved in the first case and disease improvement in the second one. Both patients were off immunosuppressive or immunomodulating therapy throughout the post-transplant period. Notably, HDCT + ASCT resulted in an excellent quality of life (QoL) response in both cases. Our findings demonstrate that HDCT + ASCT could be considered as an effective treatment for MS patients. Moreover, QoL measurement seems to be an effective approach to assessment of treatment outcomes at long-term follow-up of patients with MS.

HLA‐DRB1*16‐restricted recognition of myeloid cells, including CD34+ CML progenitor cells

Summary. The therapeutic effect of a human leucocyte antigen (HLA)‐identical allogeneic stem cell transplantation (allo‐SCT) for the treatment of haematological malignancies is mediated partly by the allogeneic T cells that are administered together with the stem cell graft. Chronic myeloid leukaemia (CML) is particularly sensitive to this graft‐versus‐leukaemia (GVL) effect. Several studies have shown that in allogeneic responses both CD4 and CD8 cells are capable of strong antigen‐specific growth inhibition of leukaemic progenitor cells, but that CD4 cells mainly exert the GVL effect against CML. Efficient activation of allogeneic CD4 cells, as well as CD8 cells, may explain the sensitivity of CML cells to elimination by allogeneic T cells. Identification of the antigens recognized by CD4 cells is crucial in understanding the mechanism through which CML cells are so successful in activating allogeneic T cells. In the present report, we describe the characterization of an allogeneic CD4 T‐cell clone, DDII.4.4. This clone was found to react against an antigen that is specifically expressed in myeloid cells, including CD34+ CML cells. The antigen recognition is restricted by HLA‐DRB1*16. To our knowledge, this is only the second report on an allogeneic CD4 T‐cell clone that reacts with early CD34+ myeloid progenitor cells.