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Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients. The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant T cells enhance proliferation of the malignant cells in an SE- and MHC class II–dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4+ T-cell lines also enhance proliferation of the malignant cells. The growth-promoting effect depends on direct cell-cell contact and soluble factors such as interleukin-2. In conclusion, we demonstrate that SE triggers a bidirectional cross talk between nonmalignant T cells and malignant CTCL cells that promotes growth of the malignant cells. This represents a novel mechanism by which infections with SE-producing bacteria may contribute to pathogenesis of CTCL.

Reduced levels of somatic hypermutation (SHM) have recently been described in IgG-switched immunoglobulin genes in a minority of patients with common variable immunodeficiency (CVID), demonstrating a disruption of the normal linkage between isotype switch and SHM. To see if, irrespective of isotype, there is a tendency to use unmutated immunoglobulin genes in CVID, we studied SHM in κ light-chain transcripts using a VκA27-specific restriction enzyme-based hot-spot mutation assay (IgκREHMA). Hot-spot mutations were found in 48% (median; reference interval, 28%-62%) of transcripts from 53 healthy controls. Values were significantly lower in 31 patients (median, 7.5%; range, 0%-73%; P < .0000001) of whom 24 (77%) had levels below the reference interval. Low levels of SHM correlated with increased frequency of severe respiratory tract infection (SRTI; P < .005), but not with diarrhea (P = .8). Mannose-binding lectin (MBL) deficiency also correlated with SRTI score (P = .009). However, the correlation of SHM and SRTI was also seen when only patients with normal MBL genotypes were analyzed (n = 18, P = .006). A slight decline of mutated fractions over years was noted (P = .01). This suggests that most patients with CVID fail to recruit affinity-maturated B cells, adding a qualitative deficiency to the quantitative deficiency characterizing these patients.

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon γ (IFN-γ)–inducible protein 10 (γIP-10) and monokine induced by IFN-γ (Mig). We report the novel finding that CXCR3 is also expressed on CD34+ hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34+ progenitors. Freshly isolated CD34+progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase–polymerase chain reaction technique. γIP-10 and Mig induced chemotaxis of GM-CSF–stimulated CD34+ progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block γIP-10–induced and Mig-induced CD34+ progenitor chemotaxis. These chemotactic attracted CD34+ progenitors are colony-forming units—granulocyte-macrophage. γIP-10 and Mig also induced GM-CSF–stimulated CD34+ progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of γIP-10 and Mig but not of chemokine stromal cell–derived factor 1α. γIP-10–induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF–stimulated CD34+progenitors. Moreover, γIP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF–stimulated CD34+progenitors. These results indicate that CXCR3–γIP-10 and CXCR3–Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34+ hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34+ hematopoietic progenitors.

Purpose: From May 2003 to March 2005, 37 patients with haematological malignancies were transplanted with peripheral blood stem cells after nonmyeloablative conditioning with fludarabine 30 mg/m2 i.v. once daily on day −4 to −2 and 200 cGY of total body irradiation on day 0. Post-transplant immunosuppression consisted of oral cyclosporin 12.5 mg/kg/day and mycophenolate mofetil 30 mg/kg/day. Three patients were not eligible for follow-up due to lack of informed consent or informative markers for chimerism measurement. The purpose of this study was to evaluate kinetics of early chimerism of CD4+, CD8+, and, CD15+ cells immediately post transplant and furthermore to identify factors associated with rejection of graft. Methods: Blood samples were collected post transplant on day +1–3, +5–7, and then weekly. Samples were separated with immunomagnetic beads and DNA was salt extracted. Chimerism analysis was performed by automated high-resolution real-time quantitative PCR based on short insertion or deletion polymorphisms or single nucleotide polymorphisms. Results: The kinetics of CD15+ chimerism differed from that of CD4+ and CD8+ chimerism (Figure). CD15+ donor chimerism was low with a median of 0.9% donor cells on day +1–3, and remained low until around day +14. Subsequently the donor chimerism increased rapidly towards 100%. The CD4+ and CD8+ donor chimerism in contrast started higher with a median of 16.3% and 15.1% respectively on day +1–3, increased slowly, and took months to reach 100%. CD4+, CD8+ or CD15+ donor chimerism on day +1–3 did not predict later development of acute graft versus host disease. Two patients had primary rejection of the donor stem cells, while four patients had secondary rejection. The patients with primary rejection had no measurable donor chimerism in any cell lineages from day +1–3. No patient with CD15+ donor chimerism above the median (0.9%) on day +1–3 rejected, while 40% (6/15) of the patients with CD15+ donor chimerism below the median rejected (LogRank test, p=0.01). No differences were found in total number of nucleated cells, CD34+, CD3+, CD4+, or CD8+ cells in the donor harvest between patient who rejected and those who did not. Conclusions: The kinetics of early CD15+ donor chimerism differed from the kinetics of early CD4+ and CD8+ donor chimerism. Early CD15+ donor chimerism was markedly lower than CD4+ and CD8+ donor chimerism. CD15+ donor chimerism increased toward 100% donor chimerism more rapidly than the CD4+ and CD8+ chimerism. Primary rejection of donor stem cells can be identified early, as these patients have no measurable donor CD4+ or CD8+ chimerism on day +1–3. Furthermore, patients with CD15+ donor chimerism above the median on day +1–3 have a low risk for rejection. Figure Figure

Cytokines are thought to play an important role in the pathophysiology of Graft-versus-Host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNC) from 21 patients with hematologic malignancies and their human leukocyte antigen identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNC were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNC of recipient, donor or HLA-mismatched third-party origin. The gene expression of interleukin (IL)-2, IL-4, IL-10, IL-18, tumor necrosis factor-α and transforming growth factor-β in each culture was then measured by real-time quantitative RT-PCR. The composition of the responder MNC differed between patients and donors and changed following HCT with a possible influence on the results. Early after transplant (day +14) the IL-10 mRNA level in response to recipient or donor antigens was higher in patients who did not develop clinical significant acute GVHD when compared to the level in patients who subsequently developed acute GVHD grades II–IV (p=0.005 and p=0.004, respectively). The IL-10 mRNA level on day +14 was highly correlated to the pre-transplant mRNA level of the recipient MNC but not to the level of the donor MNC, suggesting that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNC obtained pre-transplant from recipients who progressed or relapsed after the transplant when compared to the level in patients who did not, p=0.03. In conclusion we find that detection of cytokine gene expression in MNC by real-time quantitative RT-PCR is an interesting tool in the study of the immune responses following HCT. A number of factors are likely to influence the results obtained and in this study we have identified the composition of the MNC as such a factor. Our results suggest a role for IL-10 as an inhibitor of alloreactivity following nonmyeloablative HCT. This inhibition may have dual effects by limiting the degree of acute GVHD and at the same time increase the probability of relapse.