The role of suppressor cells in the pathogenesis of common variable hypogammaglobulinemia and the immunodeficiency associated with myeloma.

The role of suppressor cells in the pathogenesis of immunodeficiency was analyzed using a technique that permits study of the differentiation of B lymphocytes into immunoglobulin-synthesizing plasma cells. Lymphocytes from normals synthesized 4,910 ng of IgM, 1,270 ng of IgA, and 1,625 ng of IgG per 2 X 10(6) cells when cultured for 7 days in the presence of pokeweed mitogen. In contrast the lymphocytes from patients with common variable hypogammaglobulinemia did not synthesize significant quantities of immunoglobulin. When lymphocytes from 9 of 13 patients with common variable hypogammaglobulinemia studied were cocultured with normal lymphocytes, the synthesis of immunoglobulin by the normal lymphocytes was depressed by 75-100%. A comparable suppression of immunoglobulin synthesis by normal lymphocytes was observed when they were cocultured with T cells from hypogammaglobulinemic patients. These studies suggest that in some patients the disease common variable hypogammaglobulinemia may not be due to an intrinsic defect of B cells alone but may be cuased or perpetuated by an abnormality of regulatory T cells that act to suppress B-cell maturation and antibody production. Peripheral blood lymphocytes from myeloma patients also had a drastically reduced capacity to produce polyclonal immunoglobulins. Three of 6 myeloma patients tested had circulating mononuclear cells that suppressed immunoglobulin production by cocultured normal lymphocytes. Purified T cells from myeloma patients did not mediate this suppressor effect. These observations suggest that one mechanism for the humoral immune deficiency observed in myeloma patients is a block of polyclonal B-cell maturation by suppressor cells.     

Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.     

alpha-Fetoprotein values in monkey (Macaca mulatta) pregnancy.

The pregnant rhesus monkey's (Macaca mulatta) potential as a model for understanding the dynamics of alpha-fetoprotein (AFP) metabolism in human pregnancy was evaluated. AFP levels in maternal and fetal serum and amniotic fluid were determined by radioimmunoassay. Significant correlations were found between decreasing maternal serum, fetal serum and amniotic fluid AFP concentrations and increasing gestational age. However, these data are not consistent with the AFP changes reported in human pregnancy. It appears that this animal has limited applicability as a model in this aspect of human pregnancy.     

Induction of regulatory T cells and dominant tolerance by dendritic cells incapable of full activation

Transplants tolerated through a process known as infectious tolerance evoke continuous recruitment of regulatory T (Treg) cells that are necessary to maintain the unresponsive state. This state is maintained long-term and requires continuous Ag exposure. It is not known, however, whether infectious tolerance operates through sustained recruitment of pre-existing regulatory cells, induction of regulatory cells, or both. Using mice deficient in natural Treg cells, we show here that quiescent donor dendritic cells (DC) laden with histocompatibility Ag can induce Treg cells de novo that mediate transplantation tolerance. In contrast, fully activated DC fail to do so. These findings suggest that DC incapable of delivering full activation signals to naive T cells may favor their polarization toward a regulatory phenotype. Furthermore, they suggest a role for quiescent endogenous DC in the maintenance of the tolerant state.     

Involvement of IL-15 in the pathogenesis of human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis: implications for therapy with a monoclonal antibody directed to the IL-2/15R beta receptor.

Human T lymphotropic virus type I (HTLV-I) is the causative agent of an inflammatory neurological disease termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). An ongoing lymphocyte activation exists in patients with HAM/TSP, which was demonstrated by the spontaneous proliferation of their PBMC ex vivo. It was shown that spontaneous proliferation present in HAM/TSP is due, in part, to an IL-2/IL-2R autocrine loop. However, addition of Abs against IL-2 or IL-2R alpha only partially inhibited the spontaneous proliferation. Since IL-15 is a cytokine with similar functional characteristics to those of IL-2, we reasoned that IL-15 might be an additional growth factor that contributes to the spontaneous proliferation observed in HAM/TSP. In this study, we demonstrated that IL-15 mRNA expression was elevated in PBMC obtained from HAM/TSP patients when compared with those of the normal donors. Furthermore, we showed that the addition of blocking Abs against IL-15 or its receptor inhibited the spontaneous proliferation of HAM/TSP PBMC. Addition of Abs directed toward both IL-15 and IL-2, or their receptors, inhibited the proliferation almost completely. These data suggest the existence of two autocrine loops involving IL-15/IL-15R and IL-2/IL-2R, both contributing to the spontaneous proliferation of HAM/TSP PBMC.     

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman’s Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y⁴⁸¹EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y⁴⁸¹EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y⁴⁸¹EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.