Levels of CMV specific CD4 T cells are dynamic and correlate with CMV viremia after allogeneic stem cell transplantation

Cytomegalovirus (CMV) infection is the most frequent viral complication in patients after allogeneic stem cell transplantation. As CMV replication is tightly controlled by the cellular arm of specific immunity, the kinetics of CMV-specific T cells in association with individual reactivation episodes were prospectively analyzed in 40 allogeneic transplant recipients in a routine clinical setting and evaluated as determinant of impaired CMV control. Antigen-specific CD4 and CD8 T cells were quantified directly from whole blood using intracellular cytokine staining after specific stimulation and MHC class I multimers, respectively. Highly dynamic intraindividual changes of CMV-specific CD4 T cells were observed in patients experiencing CMV viremia. Episodes of CMV reactivation were associated with a drop of CMV-specific CD4 T cells that re-increased after viral clearance (p<0.0001). Furthermore, levels of CMV-specific CD4 T cells at the onset of viremia inversely correlated with peak viral load thereafter (p = 0.02). In contrast, CMV-peptide specific CD8 T cells did not show any association with viremia (p = 0.82). Interestingly, therapeutic dosages of cyclosporine A and corticosteroids led to a dose-dependent reduction of CMV-specific T-cell functions, indicating a causal link between intensified immunosuppressive treatment and CMV reactivation. In conclusion, levels of CMV-specific CD4 T cells inversely correlate with reactivation episodes and may represent a valuable measure to individually guide antiviral therapy after stem cell transplantation.     

Targeting properties of an anti-CD16/anti-CD30 bispecific antibody in an in vivo system

Bispecific antibodies are currently being used in clinical trials in increasing numbers in the areas of breast cancer, prostate cancer, non-Hodgkin's lymphoma and Hodgkin's lymphoma. We have previously performed two clinical trials in patients with Hodgkin's disease with an anti-CD30/anti-CD16 bispecific antibody and demonstrated a 30% response rate in a cohort of patients otherwise resistant to standard therapeutic modalities. However, no surrogate marker could be defined in these trials indicative of optimal antibody dosing/scheduling or predictive for favorable response. In order to evaluate accurately the potential biodistribution properties of bispecific antibody in patients, we have performed a detailed analysis of the binding properties and animal model in vivo characteristics of these constructs. For this purpose, the parental antibodies (anti-CD30 and anti-CD16) and the bispecific antibody (anti-CD30/anti-CD16) were radiolabeled with either ¹²⁵I or ¹¹¹In. Antibody integrity and binding properties after labeling were confirmed by Scatchard plot and Lindmo analysis. ¹¹¹In-labeled antibodies revealed superior targeting properties in a standard SCID mouse tumor model. Both the bivalent parental anti-CD30 monoclonal antibody and the monovalent anti-CD30/anti-CD16 bispecific antibody showed excellent uptake in CD30⁺ tumors which did not differ significantly between the two (maximum uptake 16.5% ± 4.2% vs. 18.4% ± 3.8% injected dose/gram tissue). The equivalent targeting properties of the bispecific antibody compared with the parental anti-CD30 antibody encourages the further clinical development of this bispecific antibody, and might help to explain the clinical responses seen with this antibody so far in patients suffering from Hodgkin's disease. Received: 26 October 2000 / Accepted: 15 December 2000     

Accumulation of DNA damage in hematopoietic stem and progenitor cells during human aging

Background

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of aging. Here we tested this hypothesis in healthy individuals of different ages by examining unrepaired DNA double-strand breaks (DSBs) in hematopoietic stem/progenitor cells matured in their physiological microenvironment.

Methodology/Principal Findings

To asses DNA damage accumulation and repair capacities, γH2AX-foci were examined before and after exposure to ionizing irradiation. Analyzing CD34+ and CD34− stem/progenitor cells we observed an increase of endogenous γH2AX-foci levels with advancing donor age, associated with an age-related decline in telomere length. Using combined immunofluorescence and telomere-fluorescence in-situ hybridization we show that γH2AX-foci co-localize consistently with other repair factors such as pATM, MDC1 and 53BP1, but not significantly with telomeres, strongly supporting the telomere-independent origin for the majority of foci. The highest inter-individual variations for non-telomeric DNA damage were observed in middle-aged donors, whereas the individual DSB repair capacity appears to determine the extent of DNA damage accrual. However, analyzing different stem/progenitor subpopulations obtained from healthy elderly (>70 years), we observed an only modest increase in DNA damage accrual, most pronounced in the primitive CD34+CD38−-enriched subfraction, but sustained DNA repair efficiencies, suggesting that healthy lifestyle may slow down the natural aging process.

Conclusions/Significance

Based on these findings we conclude that age-related non-telomeric DNA damage accrual accompanies physiological stem cell aging in humans. Moreover, aging may alter the functional capacity of human stem cells to repair DSBs, thereby deteriorating an important genome protection mechanism leading to exceeding DNA damage accumulation. However, the great inter-individual variations in middle-aged individuals suggest that additional cell-intrinsic mechanisms and/or extrinsic factors contribute to the age-associated DNA damage accumulation.     

Analysis of the antibody repertoire of lymphoma patients

Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkin's lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6x10(6) clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.     

Effect of neuraminidase treatment on serum reactivity to autologous leukemic blast cells

Summary The sera of 35 patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) were tested for reactivity against cell surface antigens of autologous leukemic blast cells by protein A assay (PA), immune adherence assay (IA), and anti-C3 mixed hemadsorption assay (C3-MHA). Autologous serum reactivity was detectable by PA in four cases and by LA and C3-MHA in about half the patients. Autologous serum reactivity occurred more often in ALL than in ANLL. Absorption studies revealed that in one patient only the autologous reactivity was directed against a restricted antigen, which could be detected only on the individual T-ALL blast cells. All other autologous antibodies detected unspecific antigens. Neuraminidase treatment had two effects: first, it increased antibody attachment to antigens which are also present on untreated cells; secondly, after neuraminidase treatment an antigen was detectable on the cell surface which could also be demonstrated on neuraminidase-treated non-leukemic cells (e.g., erythrocytes). Neither of these two effects of neuraminidase treatment seems to be tumor-specific. Possible therapeutic effects of neuraminidase are probably caused by unspecific adjuvant effects of the enzyme.     

Targeted bioactivity of membrane-anchored TNF by an antibody-derived TNF fusion protein

We describe the generation and characterization of a fusion protein consisting of a humanized anti-fibroblast-activating protein (anti-FAP) Ab and human TNF replacing the IgG1 CH2/CH3 Fc domain. The construct was generated by recombinant DNA technology and preserved its IgG1-derived dimeric structure with the TNF molecule linked as a dimer. Expression in CHO cells was optimized in serum-free medium under GMP conditions to achieve production levels up to 15 mg/liter. Recognition of the FAP Ag by the construct was as good as that by the parental anti-FAP Ab. TNF signaling was induce able via both TNF receptor types. When acting in solution, the Ab-linked TNF dimer exhibited a 10- to 20-fold lower activity compared with recombinant trimeric TNF. However, after binding to FAP-expressing cells, immobilized anti-FAP-TNF dimer was equivalent to membrane-anchored TNF with regard to bioactivity. Amplification of TNF-related pathways by mimicking the membrane-integrated TNF signaling was detectable in various systems, such as apoptosis induction or tissue factor production. The difference in TNF receptor type 1 and 2 signaling by the anti-FAP-TNF construct correlated well with its Ag-bound or -soluble status. Translating the approach into a xenograft animal model (BALB/c nu/nu mice), we demonstrated low toxicity with measurable antitumor efficacy for the TNF fusion protein after i.v. application. Immunohistochemical analysis of tumor sections showed restricted TNF-mediated macrophage recruitment to the targeted tissue in a time- and dose-dependent manner. These data warrant transfer of the anti-FAP-TNF immunocytokine into clinical trials for the treatment of FAP-positive tumors.     

B lymphocyte involvement in ankylosing spondylitis: the heavy chain variable segment gene repertoire of B lymphocytes from germinal center-like foci in the synovial membrane indicates antigen selection

The synovial membrane (SM) of affected joints in ankylosing spondylitis (AS) is infiltrated by germinal center-like aggregates (foci) of lymphocytes similar to rheumatoid arthritis (RA). We characterized the rearranged heavy chain variable segment (VH) genes in the SM for gene usage and the mutational pattern to elucidate the B lymphocyte involvement in AS.     

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.