Search

Tertiary lymphoid structures (TLS) form at sites of chronic inflammation, and locally prime the immune system independently of secondary lymphoid organs. The intratumoral presence of TLS has been associated with a positive prognostic outcome for patients with solid tumors, including colorectal carcinoma. Thus, the therapeutic induction of TLS would be hypothesized to confer clinical benefit to patients. Indeed, the therapeutic introduction of the IL-1 family cytokine IL-36 gamma into the tumor microenvironment (TME) of murine MC38 colon carcinoma leads to delayed tumor progression and the development of TLS at 5 days post-treatment. The TLS present with characteristics of secondary lymphoid organs, including PNAd+ high endothelial venules (HEV) surrounded by CD3+ T cells and CD11c+ dendritic cells, but are considered as “non-classical” due to the absence of a defined B cell zone. The formation of TLS is dependent upon the adaptive immune response, as IL-36 gamma-treated tumors grown in Rag2−/− animals fail to present with PNAd+ vasculature within the tumor. Thus, crosstalk between the immune system and cells of the vasculature is key to the efficacy of the IL-36 gamma-induced anti-tumor immune response. In human colorectal carcinoma, IL-36 gamma was also observed to promote the local anti-tumor immune response. Expression of IL-36 gamma by vascular endothelial cells of HEV was associated with an increased density of CD20+ B cells in TLS. Specific IL-36 gamma production by CD68+ macrophages was associated with an increased infiltration of CD4+ central memory T cells into the TME. Therefore, IL-36 gamma is a pro-inflammatory mediator involved in the anti-tumor immune response in both murine and human colorectal cancer.

Immune responses are initiated by dendritic cells (DC) that form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells colonizing epithelial surfaces. We have recently shown that macrophage-inflammatory protein-3α/CCL20, a chemokine secreted by epithelial cells, induces the selective migration of LC among DC populations. In this study, we investigated the effects of cytokines on the expression of the CCL20 receptor, CCR6, during differentiation of LC. We found that both IL-4 and IFN-γ blocked the expression of CCR6 and CCL20 responsiveness at different stages of LC development. The effect of IL-4 was reversible and most likely due to the transient blockade of LC differentiation. In contrast, IFN-γ-induced CCR6 loss was irreversible and was concomitant to the induction of DC maturation. When other cytokines involved in DC and T cell differentiation were tested, we found that IL-10, unlike IL-4 and IFN-γ, maintained CCR6 expression. The effect of IL-10 was reversible and upon IL-10 withdrawn, CCR6 was lost concomitantly to final LC differentiation. In addition, IL-10 induced the expression of CCR6 and responsiveness to CCL20 in differentiated monocytes that preserve their ability to differentiate into mature DC. Finally, TGF-β, which induces LC differentiation, did not alter early CCR6 expression, but triggered its irreversible down-regulation, in parallel to terminal LC differentiation. Taken together, these results suggest that the recruitment of LC at epithelial surface might be suppressed during Th1 and Th2 immune responses, and amplified during regulatory immune responses involving IL-10 and TGF-β.

We demonstrate that soluble CD16 (sCD16; soluble FcγRIII), a natural ligand of CR3, inhibits the infection of monocytes by primary R5 HIV-1 strain opsonized with serum of seronegative individuals. Inhibition of monocyte infection by sCD16 was similar to that observed with anti-CR3 mAbs, indicating that opsonized HIV may use a CR3-dependent pathway for entry in monocytic cells. Cultured human monocytes express both CR3 (CD11b/CD18) and CCR5 receptors. RANTES, the natural ligand of CCR5, inhibited infection of monocytes with unopsonized HIV particles and partially that of monocytes infected with HIV particles opsonized with complement-derived fragments. Although HIV-infected monocytes from homozygous CCR5 Δ 32/Δ 32 (CCR5−/−) individuals produce low levels of p24, cells infected with opsonized particles produced higher levels of p24 than cells infected with unopsonized particles. Our results thus suggest that CR3 may represent an alternative coreceptor to CCR5 of opsonized primary R5 virus entry into monocytes/macrophages. We also observed that the concentration of sCD16 is greatly decreased in sera of HIV-infected patients with low lymphocyte CD4+ counts. Taken together, our findings suggest that sCD16, present in plasma, may play an important role in controlling HIV-1 spread.

Immune complex-mediated inflammatory responses are initiated by FcγR on phagocytes. We report in this study that an inhibitory receptor, FcγRIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory FcγR it down-regulates effector function. FcγRIIb2 expression is increased by IL-4 and decreased by IFN-γ, in contrast to the activating receptor, FcγRIIa, which is increased by IFN-γ and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing FcγR systems, altering the balance of activating and inhibiting FcγR. The detection and cytokine modulation of FcγRIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.

FcγRIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They are widely expressed by cells of hematopoietic origin. We investigated here whether FcγRIIB could also negatively regulate protein tyrosine kinase receptor (RTK)-dependent cell proliferation. As an experimental model, we used growth factor-dependent mast cells that constitutively express FcγRIIB and c-kit, an RTK prototype. We found that anti-c-kit Abs mimicked the effect of stem cell factor and induced thymidine incorporation in FcγRIIB−/−, but not in wild-type (wt) mast cells unless FcγRIIB were blocked or anti-c-kit F(ab′)2 were used. When coaggregated with c-kit by intact Abs in wt mast cells, FcγRIIB inhibited thymidine incorporation, as well as cell proliferation, and inhibition was correlated with an arrest of cells in G1 during the cell cycle. The coaggregation of c-kit with FcγRIIB did not affect ligand-induced c-kit phosphorylation and induced the tyrosyl-phosphorylation of FcγRIIB, which selectively recruited the Src homology 2 domain-bearing inositol 5-phosphatase SHIP. Our results indicate that IgG Abs to growth factors or growth factor receptors may control RTK-dependent proliferation of a variety of cells that express FcγRIIB.

FcγRIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunoreceptor tyrosine-based inhibition motif (ITIM). FcγRIIB inhibit cell activation triggered by receptors that signal via immunoreceptor tyrosine-based activation motifs. This inhibition requires ITIM tyrosyl phosphorylation and is correlated with the binding of SH2 domain-containing phosphatases that may mediate the inhibitory signal. In the present work, we investigated the mechanism of FcγRIIB phosphorylation and its consequences in mast cells. We demonstrate that the phosphorylation of FcγRIIB requires coaggregation with FcεRI and that, once phosphorylated, FcγRIIB selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in vivo. In vitro, however, the phosphorylated FcγRIIB ITIM binds not only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and SHP-2. We show that the coaggregation of FcγRIIB with FcεRI does not prevent FcεRI-mediated activation of lyn and syk. Both kinases can phosphorylate FcγRIIB in vitro. However, when coaggregated with FcεRI, FcγRIIB was in vivo phosphorylated in syk-deficient mast cells, but not in lyn-deficient mast cells. When FcεRI are coaggregated with FcγRIIB by immune complexes, FcεRI-associated lyn may thus phosphorylate FcγRIIB. By this mechanism, FcεRI initiate ITIM-dependent inhibition of intracellular propagation of their own signals.

Lymph node lymphocytes from rats immunized with a guinea pig encephalitogenic protein (GEProt) were incubated in vitro with the immunizing antigen in a serum-free medium. They released a factor which had the property to combine with IgG antibodies complexed with antigen, but not IgM antibodies, inducing hemagglutination of IgG-sensitized erythrocytes and protecting IgG-sensitized erythrocytes, but not IgM-sensitized erythrocytes from complement-induced hemolysis. A similar activity was found in supernatants from mixed leukocyte cultures between allogenic mouse strains. This factor was named immunoglobulin-binding factor (IBF). IBF was separated in rat from migration inhibitory factor, blastogenic factor, and lymphotoxin upon column fractionation techniques. IBF activity was found in fractions of about 150,000 m.w. and with a pH of about 6.30.

A subpopulation of activated murine T cells (25%) were found to bear Fc receptors for mouse, rabbit, and human IgG. Studies, by rosette inhibition and indirect immunofluorescence, of their specificity for human IgG subclasses have shown that these receptors bound nonaggregated IgG1, IgG2, and IgG3 in a comparable fashion, but did not bind IgG4. The binding site has been localized exclusively to the Fc region and the binding capacity of this fragment was not affected by mild reduction. Binding of the Fc fragment of IgG4 could not be demonstrated. Using isolated Cγ2 and Cγ3 (pFc′) domains from IgG1 and a Cγ3-derived fragment (Fc′), a major binding site was localized to the Cγ3 domain between residues Gln342 and His433. A 10-fold molar excess of Cγ3 dimer over native Fc was, however, required to show comparable activity. The monomeric Cγ2 domain was 10-fold less active than the Cγ3 fragment. Human urine β2-microglobulin was not cytophilic. These data are discussed and it is suggested that the Cγ2 and Cγ3 domains may contribute to the formation of a cooperative binding site through quaternary interdomain interactions. By redistribution experiments, it was demonstrated that the three human IgG subclasses shown to be cytophilic share common or linked receptors, which were not associated with the H-2 alloantigens.

L-5178-Y thymoma cells were used to produce radioactive immunoglobulin-binding factor (IBF). For this purpose, the cells were internally labeled by incubation with radioactive amino acids and/or fucose. The supernatants contained radioactive material that bound to IgG-sensitized erythrocytes and suppressed the in vitro antibody response to sheep red blood cells. Upon filtration on Sephadex G-200 both the IgG-binding activity and the suppressive activity eluted at peaks of 140,000 and above 300,000 d. However, on SDS polyacrylamide gels, after precipitation with antigen-IgG-antibody complexes, IBF was found in a single peak of 80,000 d. This molecule could be dissociated in the presence of mercaptoethanol into a major unit of 40,000 d and a minor unit of 20,000 d. These data suggest that IBF is a molecule of 80,000 d, which contains chains of 40,000 d and probably 20,000 d linked by disulfide bridges. In cell supernatants, however, the factor exists in polymeric forms of 140,000 d and more than 300,000 d.

Purified peripheral blood lymphocytes from 13 healthy donors, 6 melanoma patients and 1 halo nevus patient were tested for cytotoxic activity against an allogeneic melanoma cell line (IGR3) in, at least, one of the following assays: cell-mediated cytotoxicity (CMC), antibody-dependent cellular cytotoxicity (ADCC) and microcytotoxicity assays (MA). The lymphocytes were isolated by Ficoll-Triosil gradient centrifugation (fraction F) followed by removal of iron-phagocytosing and adherent cells (fraction FFF) and by subsequent passage through anti-IgG columns (fraction FFF-C). Leukocytes of each fraction were identified by different methods including morphology, rosetteformation, phagocytic activity, and membrane fluorescence. CMC activity paralled ADCC activity at a log lower level of sensitivity. In both assays lymphocytes of fractions F and FFF had the highest activity, whereas in fraction FFF-C cytotoxicity was strongly reduced. In all three lymphocyte fractions CMC and ADCC activity could be blocked by preincubation of the effector cells in aggregated IgG. Furthermore, depletion of E rosette-forming lymphocytes slightly increased ADCC and CMC activity, whereas depletion of EA and EAC rosette-forming lymphocytes strongly decreased it. Our results therefore indicate that in both CMC and ADCC assays, non-adherent, non-phagocytic Fc receptor-bearing lymphocytes (“K” cells) were the active cytotoxic cells. In MA, on the other hand, mononuclear phagocytes seemed to be the most active cell population. So far no significant difference was observed in CMC, ADCC, and MA between control persons and melanoma patients.

“Sensitized” lymphocytes from rats, mice, and human beings were cultured in the presence of the immunizing antigen for 24 hr in a serum-free system. Under these conditions the lymphocytes released a soluble factor which prevented the combination of C1 with antigen-IgG complexes. This factor prevented the fixation of guinea-pig C1, as well as the fixation of rat, human and hamster C1. The fixation of C1 on antigen-IgM complexes was not blocked. After purification of this factor from rat lymphocytes, it was shown to have a m.w. of about 150,000, a sedimentation constant of 7S, an electrophoretic mobility of a γ-globulin and an isoelectric point of 6.30. It was remarkably stable both at 56°C and over a wide range of pH values. When used either in a soluble purified state or as a complex with antigen-IgG antibody it did not combine with non-aggregated IgG in the fluid phase, nor did it destroy C1 in serum.