Characterization of T cell repertoire in patients with graft-versus-leukemia after donor lymphocyte infusion.

The clinical efficacy of donor lymphocyte infusions (DLI) in patients with relapsed chronic myelocytic leukemia after allogeneic bone marrow transplantation has been demonstrated in several recent studies. Although it is presumed that allogeneic T cells mediate this graft-versus-leukemia (GVL) effect, the influence of DLI on the T cell compartment of recipients has not been determined. To characterize the immunologic effects of DLI and to identify T cell changes selectively associated with the GVL response, we analyzed the T cell receptor (TCR) repertoire in four patients with relapsed chronic myelocytic leukemia who achieved a complete remission after infusion of CD4+ lymphocytes from HLA-identical sibling donors. Only one of the four patients developed clinically significant graft-versus-host disease (GVHD) after infusion of donor lymphocytes. TCR repertoire was examined after PCR amplification of 24 Vbeta gene subfamilies in serial samples obtained over a 1-yr period before and after DLI. Results were compared to 10 normal donors. Before DLI, all four patients were found to have abnormal TCR Vbeta repertoire in peripheral T cells, associated with a large number of clonal and oligoclonal patterns. Abnormal TCR patterns persisted for at least 3 mo after DLI, but thereafter gradually began to normalize. By 1 yr after DLI, all patients demonstrated almost complete normalization of Vbeta repertoire with polyclonal representation within almost all Vbeta gene subfamilies. We also examined changes in the TCR Vbeta repertoire associated with the disappearance of Ph+ cells. In each patient, we were able to identify the expansion of at least 1 Vbeta gene subfamily that coincided with the time of the cytogenetic response. In one patient who was studied in greater detail, CDR3 size analysis of serial samples after DLI indicated that these changes were associated with the appearance of clonal T cells. This finding was confirmed through CDR3 sequence analysis and use of CDR3 clone-specific oligonucleotide probes. A putative GVL clone identified by this technique was not detectable in either donor or patient T cells before DLI, but persisted in peripheral T cells for approximately 1 yr. These experiments therefore provide evidence for the clonal expansion of allogeneic T cells that may be selective mediators of antileukemia activity without also mediating graft-versus-host disease.

Interleukin 17, a T-cell-derived cytokine, promotes tumorigenicity of human cervical tumors in nude mice.

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.

Accumulation of T-cell clones producing high levels of both granulocyte-macrophage and macrophage colony-stimulating factors (CSF-1) in lymph nodes involved by Hodgkin's disease.

We have previously shown that total T cells derived from lymph nodes (LN) involved by Hodgkin's disease (HD) secrete higher levels of colony-stimulating activity than total T cells present within benign hyperplastic (BH) LN and B-non-Hodgkin's lymphoma (B-NHL) LN, suggesting that T cells with particular properties accumulate in HD LN. To further characterize this T-cell population, we have quantified production of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) production in a total of 98 T-cell clones (TCC) derived from CD25+ activated T cells present in HD LN; TCC derived from CD25+ T cells obtained from B-NHL LN(101 TCC), BH LN(95 TCC), and peripheral blood (PBL; 38 TCC) of healthy donors were used as controls. HD LN were characterized by the presence of an elevated number (44%) of TCC producing particularly high titers of both GM-CSF and M-CSF, whereas only a minority of such TCC was found in control groups (10% in B-NHL, 16% in BH, 8% in PBL). These observations support the hypothesis of a selection of T-cell families with particular properties occurring in contact with Reed-Sternberg (RS) cells. According to the biological properties of GM-CSF and M-CSF, it seems reasonable to suggest the involvement of this particular subset of T cells in the granulomatous process, the peripheral blood polynucleosis, and in the paracrine growth of RS cells.

Impaired clonogenic potential of CD25 positive T cells in lymph nodes involved by B cell non-Hodgkin's lymphomas.

In vivo activated T cells (CD25+) present in lymph nodes involved by B-non-Hodgkin's lymphomas (B-NHL) were investigated here for their ability to proliferate in vitro. CD25-/CD25+ T cells were isolated using a rosette method with magnetic beads, then the frequency of proliferating T lymphocyte-precursors (PTL-P) in both populations was assessed by limiting dilution experiments, in the presence of IL2, PHA and allogeneic spleen cells as feeders. In a total of 16 cases studied, growing microcultures were observed in all cases for CD25- T cells (mean value of PTL-P frequency: 1/32; range 1/10 - 1/2899) but in 6 cases only for CD25+ T cells (mean value of PTL-P frequency: 1/441; range 1/119 - 1/3736); the absence of any proliferative cultures in the 10 other cases indicated that the number of PTL-P was inferior to 1/12480. These results suggest that the proliferative potential of CD25+ T cells infiltrating lymph nodes involved by B-NHL is paradoxically decreased.

Valid estimation of IL2 secretion by PHA-stimulated T-cell clones absolutely requires the use of anti-CD25 monoclonal antibody to prevent IL2 consumption.

A major problem encountered for quantification of IL2 production by stimulated T cells is its simultaneous consumption by these activated cells. In the present study, 40 T-cell clones (TCC) derived from normal peripheral blood, hyperplastic lymph nodes (LN) or lymph nodes involved by malignant lymphomas, were studied for their ability to produce IL2. When supernatants were generated in the presence of 20% fetal calf serum (FCS), no IL2 could be detected for 22 of the 40 TCC, whereas very low levels were found for the 18 other TCC (mean value 31 pg/ml; range from 10 pg/ml to 114 pg/ml); in contrast, when conditioned media were produced with reduced amounts of FCS (final concentration, 1%) as well as in the presence of an anti-CD25 monoclonal antibody (final concentration, 50 micrograms/ml), all TCC were found to release IL2, and very high quantities of this lymphokine were measured (mean value: 11,387 pg/ml; range, from 250 pg/ml to 37,000 pg/ml). Consequently, inhibition of IL2 consumption by PHA-stimulated TCC seems to be an absolute requirement for estimating the true capacity of T cells to produce this lymphokine.

Detection, isolation and functional studies of CD25+ T cells in lymph nodes involved by B-cell non-Hodgkin's lymphomas.

We searched for the presence of IL2 receptor (CD25) on T cells as an activation marker in lymph nodes involved by B-cell non-Hodgkin's lymphomas (B-NHL). In 26 malignant lymph nodes studied, the number of CD25+ T cells among total T cells was usually low when assessed by immunofluorescence analysis (mean +/- SD: 6.7% +/- 11.2%), but greatly increased when an immunomagnetic rosette method was used (mean +/- SD: 17.5% +/- 16.6%). In six cases, CD25-/CD25/CD25+ cells were isolated by immunomagnetic separation, with a purity greater than 97% for both populations. Expansion of CD25-/CD25+ T cells was obtained with IL2 and PHA, then conditioned media (CM) were prepared. No IL2 activity was found in CM from both CD25-/CD25+ T cells when tested on CTLL2 cells. BCGF and BCDF mu/gamma activities were assayed on normal B cells stimulated with soluble or insolubilized anti-mu antibodies(BCGF) or with Cowan I (BCDF). Results of production of all these activities were comparable for both populations, and thus do not favour the possibility that CD25+ T cells closely associated with malignant B-NHL cells in lymph nodes may influence their proliferation (BCGF) or expression/secretion of heavy chain isotype (BCDF mu/gamma).

Clinical and biologic effects of anti-interleukin-10 monoclonal antibody administration in systemic lupus erythematosus.

OBJECTIVE: To evaluate the safety and clinical efficacy of administering an anti-interleukin-10 (anti-IL-10) monoclonal antibody (mAb) to systemic lupus erythematosus (SLE) patients with active and steroid-dependent disease. In addition, we sought to assess the effects of in vivo IL-10 neutralization on biologic markers of SLE. METHODS: Treatment consisted of 20 mg/day intravenous administration of an anti-IL-10 murine mAb (B-N10) for 21 consecutive days, with a followup period of 6 months. Six patients were studied. RESULTS: Treatment was safe and well tolerated. All patients developed antibodies against B-N10. Cutaneous lesions and joint symptoms improved in all patients beginning during B-N10 administration and continuing to month 6. The SLE Disease Activity Index decreased from a mean +/- SEM of 8.83+/-0.91 on day 1 to 3.67+/-0.67 on day 21 (P = 0.001), 1.50+/-0.84 at month 2, and 1.33+/-0.80 at month 6 (P<0.001). At the end of followup, the disease was clinically inactive in 5 of the 6 patients. Prednisone administration was decreased from a mean +/- SEM of 27.9+/-5.7 mg/day on day 1 to 9.6+/-2.0 mg/day at month 6 (P<0.005). Activity of immune and endothelial cells rapidly decreased, as assessed by the early evolution of several biologic markers. CONCLUSION: This is the first report of IL-10 antagonist administration to humans. The study shows the involvement of IL-10 in the pathogenesis of SLE, and indicates that the use of IL-10 antagonists may be beneficial in the management of refractory SLE.