Minor histocompatibility antigen-specific T cells with multiple distinct specificities can be isolated by direct cloning of IFNgamma-secreting T cells from patients with relapsed leukemia responding to donor lymphocyte infusion.

Graft-vs-leukemia reactivity after donor lymphocyte infusion (DLI) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on recipient hematopoietic cells. To study the diversity of cells involved in this immune response, hematopoietic cell reactive T cells were directly clonally isolated from peripheral blood of patients entering complete remission after DLI. T cells were briefly stimulated with bone marrow cells from patients pretransplant, and IFNgamma-secreting T cells were directly clonally isolated, and expanded. Cytotoxic T-lymphocyte (CTL) clones from individual patients used multiple distinct HLA-restricting molecules and varied in reactivity against patient-derived normal and/or malignant hematopoietic cells. For each patient, CTL clones specific for known immunodominant mHags as well as distinct unknown mHags were found. Within individual patients, CTL clones using the same HLA-restricting element could show differential recognition patterns, indicating further diversity in mHag reactivity. CTL clones from individual patients exhibiting identical specificities could show oligoclonal origin. In conclusion, the direct cloning technique shows that the response to hematopoietic cells after DLI is directed against multiple distinct mHags, including but not limited to known immunodominant mHags, implying that immunotherapy with T cells against multiple mHag specificities may be more effective in eradicating malignant cells.

[Physicians and scientific research: slight decline of the numbers of physicians with a doctoral degree]. / Artsen en wetenschappelijk onderzoek: lichte teruggang van het aantal gepromoveerde artsen.

OBJECTIVE: To establish whether the number of physicians interested in a career in academia (i.e. research) is declining. DESIGN: Descriptive. METHOD: The researchers analysed the pre- and post-doctoral careers of PhD students at 3 university medical centres (VU Amsterdam, Nijmegen and Maastricht) in 4 separate reference years (1989, 1994, 1999 and 2003), using information from doctoral dissertations and the Dutch medical address book. The researchers recorded the gender of the students and the timing of the doctorate in relation to specialist training, university education and employment, as applicable. RESULTS: The total number of dissertations produced at the 3 medical faculties in the 4 reference years increased gradually by nearly a factor of 2 (1989: 112; 1994: 152; 1999: 198; 2003: 213). In terms of absolute numbers, the number of dissertations authored by physicians increased from 1989 to 1994 and again in 1999 (64, 90 and 105), but decreased slightly in 2003 (96). The percentage of female physicians obtaining a doctorate doubled during this period (1989: 9/64 (14); 2003: 28/96 (29)). Increasingly, physicians prepared their dissertation before or during their training as specialists or general practitioners (1989: 15/64 (23%); 2003: 51/96 (53%)). Ofthe clinical specialists who had received their doctorate, approximately half continued to work in an academic setting after obtaining their degree. This percentage remained approximately the same in all reference years (1989: 13/26 (50); 1994:19/35 (54); 1999: 21/45 (47); 2003: 21/40 (53)). CONCLUSION: Although the number of physicians performing scientific research as part of their doctoral degree project declined slightly in 2003 following an initial rise, our data indicate no cause for major concern. One reason may be increased interest in Clinical Research Fellow programmes. However, the future of medical research would look brighter if young physicians with doctorates had better career prospects within academic centres. To follow the academic careers of clinicians in The Netherlands, a national registry is needed to collect the type of data analysed in this study continually.

Direct cloning of leukemia-reactive T cells from patients treated with donor lymphocyte infusion shows a relative dominance of hematopoiesis-restricted minor histocompatibility antigen HA-1 and HA-2 specific T cells.

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5-7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8+ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNgamma were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3-35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.