The role of the Fc receptor (FcR) of thymus-derived lymphocytes. II. Presence of FcR on suppressor cells and direct involvement in suppression.

Alloantigen-activated T cells (ATC) were prepared by injecting C3H/He thymocytes into host-irradiated BALB/c mice. ATC were taken from the spleen of the recipients at day 5 and tested for their activity on “In vitro” primary antibody production to sheep red blood cells (SRBC) induced in splenic C3H/He lymphocytes. ATC added 24 h after antigen were found to be suppressive. Separation of ATC by velocity sedimentation, according to cell size showed that suppressor cells were found in each population, medium and large lymphocytes, however, being more active. Separation of ATC, according to the presence of Fc receptor (FcR) at their membrane was achieved by velocity sedimentation after rosetting with IgG-sensitized SRBC (EA). Suppressor cells were then found in FcR⁺ and not in FcR^? fractions, the degree of suppression being parallel to the ratio of FcR⁺ cells. At both extremes, pure populations of FcR⁺ cells were highly suppressive, while populations of ATC, completely devoid of FcR⁺ cells, were inactive. Since FcR is a very labile molecule at the T cell surface and shed within 3 h at 37°C, but not at 4°C, ATC were pre-incubated at each temperature and added 24 h after antigen to the spleen cell cultures. In these cases, ATC having released their FcR, were much less suppressive than control ATC, and in previous work we have shown that the suppressive activity was found in the cell supernatant associated with an FcR-like molecule (immunoglobulin-binding factor). The data therefore allow the conclusion that FcR may be a marker of nonantigen-specific suppressor T cells and seems to be involved itself in the suppression phenomenon.     

The role of the Fc receptor (FcR) of thymus-derived lymphocytes. I. Presence of FcR on cytotoxic lymphocytes and absence of direct role in cytotoxicity.

Cytotoxic T lymphocytes (CTL) were prepared by injecting C3H/He (H-2^k) thymocytes into host-irradiated BALB/b (H-2^b) mice. Specific cytolysis was measured on MBL2 lymphoma cells from C57BL/6 (H-2^b) mice. Alloantigen-activated T cells (ATC) were taken from the spleens of the recipients at day 5 and separated according to size and/or to the presence of membrane Fc receptors (FcR) by the use of velocity sedimentation techniques. The data showed that CTL were found in populations of lymphocytes heterogeneous in cell size and were always present in the FcR-positive population. Depletion of FcR-positive ATC, after rosetting with IgG-sensitized sheep erythrocytes led to an abolishment of cytolytic activity while enrichment in FcR-positive ATC led to a parallel increase of cytolytic activity. Taking advantage of the rapid release of FcR at 37°C by ATC, we investigated the direct involvement of the FcR molecule in cytolysis. We found that ATC which had released their FcR after pre-in-cubation for 2 h at 37°C exerted the same level of cytotoxicity as control ATC incubated at 4 °C. The data lead us, therefore, to conclude that FcR may be a marker of differentiation of cytotoxic T cells found in the spleen, but does not itself play a role in the cytolytic reaction.     

The regulation of polyclonal immunoglobulin synthesis by FcR+ and FcR- monocyte subsets

FcR+ and FcR- monocyte subsets were added to the pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I-stimulated cultures of peripheral blood mononuclear cells (PBMC) or to PBMC depleted of monocytes. The numbers of immunoglobulin-secreting cells (ISC) and cells with intracytoplasmic immunoglobulins (PC) were evaluated 6 days later. The addition of FcR- subset increased the number of ISC in cultures of PBMC stimulated with PWM and reconstituted the response of monocyte depleted PBMC. In contrast, FcR+ monocytes suppressed PWM-induced response and, when added in high dose, also that induced by S. aureus. The FcR+ monocytes suppressed the response by inhibition of immunoglobulin secretion but not the development of PC. This suggests that FcR+ monocytes may modulate humoral response by preferential inhibition of the final differentiation of B lymphocytes into ISC.     

Receptors for immunoglobulin isotypes (FcR) on murine T cells. I. Multiple FcR expression on T lymphocytes and hybridoma T cell clones

T cell receptors for the Fc portion of the various isotypes of mouse immunoglobulins (FcR) were examined by rosette formation, using as indicator cells erythrocytes coated with monoclonal antibodies of all known isotypes of serum immunoglobulins. Three populations of mouse T cells were studied: normal thymocytes, activated T cells (ATC), generated by educating thymocytes in lethally irradiated allogeneic hosts, and hybridoma T cells, derived from somatic hybridization of ATC with the FcR-negative thymoma BW.5147. We found that many different FcR could be distinguished by their specificity for a single isotype or for a combination of several isotypes; ATC and hybridoma T cells expressed several such receptors that, at least in cloned cells, could be demonstrated to be borne by individual cells; hybridoma T cells of independent origin bore indistinguishable receptors whereas ATC expressed markedly different FcR and upon overnight incubation at 37 degrees C, immunoglobulins were found to bind onto the cell surface, even though no corresponding constitutive FcR was detected. The same was observed with hybridoma T cells and with thymocytes. It follows that a single T cell can express several FcR. Altogether, these FcR are capable of binding all known isotypes of serum immunoglobulins. They differ from one T cell to another.     

Regulatory role of FcR+ and FcR- monocyte subsets in Mycobacterium leprae-induced lymphoproliferative response in vitro.

We investigated nine rhesus monkeys (Macaca mulatta) inoculated with Mycobacterium leprae and three normal human contacts. Peripheral blood monocytes were separated into Fc receptor positive (FcR+) and Fc receptor negative (FcR-) fractions, and their regulatory role in the lymphoproliferative response in vitro to M. leprae was studied. FcR- monocytes had strong antigen presentation activity and produced no suppressor effect while FcR+ monocytes had weak antigen presentation activity and produced a non-specific suppressor factor spontaneously. With this assay system we determined that M. leprae-inoculated rhesus monkeys could be divided into three groups: good responders, very weak responders, and non-responders.     

Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses

HPK1 is a Ste20-related serine-threonine kinase that inducibly associates with the adaptors SLP-76 and Gads after T cell receptor (TCR) signaling. Here, HPK1 deficiency resulted in enhanced TCR-induced phosphorylation of SLP-76, phospholipase C-gamma1 and the kinase Erk, more-persistent calcium flux, and increased production of cytokines and antigen-specific antibodies. Furthermore, HPK1-deficient mice were more susceptible to experimental autoimmune encephalomyelitis. Although the interaction between SLP-76 and Gads was unaffected, the inducible association of SLP-76 with 14-3-3tau (a phosphorylated serine-binding protein and negative regulator of TCR signaling) was reduced in HPK1-deficient T cells after TCR stimulation. HPK1 phosphorylated SLP-76 and induced the interaction of SLP-76 with 14-3-3tau. Our results indicate that HPK1 negatively regulates TCR signaling and T cell-mediated immune responses.     

The roles of soluble osteopontin using osteopontin-transgenic mice in vivo: proliferation of CD4+ T lymphocytes and the enhancement of cell-mediated immune responses.

We generated transgenic mice expressing osteopontin (OPN) under the control of the alpha(1)-antitrypsin promoter. These mice (OPN-T mice) expressed OPN mRNA in liver and kidney, and released a large amount of plasma OPN, which increased after stimulation with turpentine oil. Before sensitization, the number of CD4+ T cells in lymph nodes was significantly higher in OPN-T than nontransgenic mice, and that in spleen was slightly higher, whereas that of CD8+ T cells was no different between OPN-T and nontransgenic mice. After sensitization, the CD4+ T cell numbers in spleen increased significantly, while there were almost no changes in the CD8+ T cells in lymph nodes and spleen. The intensity of contact hypersensitivity responses to 2,4-dinitrofluorobenzene (DNFB) was obviously enhanced in OPN-T mice. In the delayed-type hypersensitivity (DTH) model elicited by DNFB, the number of CD8+ T cells among DNFB-2,4,6-trinitrobenzenesulfonic acid (TNBS)-peritoneal exudate cells was significantly higher in OPN-T than nontransgenic mice, while there was almost no difference in that of CD4+ T cells. Adoptive transfer experiments revealed that the enhanced reactivity is carried by CD4+ and CD8+ T cells, respectively, although the ability of transferring DTH was significantly lower in CD8+ than in CD4+ T cells. The enhancement of CD8+ T cell migration was observed in OPN-T mice. These results suggest that OPN induces a proliferation of effector CD4+ and CD8+ cells in cell-mediated reactions and plays a role in the migration of CD8+ T cells.     

Immunoregulatory confluence: T cells, Fc receptors and cytokines for IgA immune responses

IgA isotype responses are regulated by at least two compartments including those of CD4+ Th2 type cells and cytokines produced by these cells. Interaction of CD4+ Th cells and APC via TCR and Ag-MHC II leads to activation of Th2 type cells. This would allow for secretion of cytokines, especially IL-5 and IL-6 which are key cytokines for the terminal differentiation of B cells into Ig secreting cells. Further, expression of Fc alpha RII on CD4+ Th2 cells could be important for the recruitment of sIgA+ B cells which would allow selective interactions of Th2 cells and sIgA + B cells via Fc alpha RII. This could lead to selectively transfer of IL-5 and IL-6 to sIgA + B cells from CD4+ Th2 cells.     

Receptors for the Fc portion of immunoglobulins (FcR) on murine oral mucosal T cells

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear Fc gamma R whereas less than 5% of T cells of any subset bear Fc alpha R or Fc mu R. In frozen tissue sections, Fc gamma R+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that Fc gamma R+ cells may be involved in the surveillance of the epithelium while the minor Fc alpha R+ L3T4+ T lymphocyte population may promote the expression of sIgA by resident sIgM-bearing B cells, and their differentiation into IgA plasma cells.     

T cell-mediated immune responses of lupus-prone BXSB mice and other murine strains.

Cellular-mediated immunity in the newly described BXSB strain of mice, which is prone to autoimmune disease, has been compared with that of two other strains, C57Bl/6 and 129/J. Quantificaiton of cytotoxic T cell responses to alloantigens and viruses (lymphocytic choriomeningitis and vaccinia virus) showed no difference in the kinetics of appearance and relative activity of cytotoxic T cells per spleen between the young and old BXSB and the control mice. The T cell-dependent primary footpad swelling after local injection with lymphocytic choriomeningitis virus was within the same range for all strains tested with respect to kinetics, but the size was greater by two-fold in C57Bl/6 mice. The susceptibility to systemic infection and subsequent induction of lymphocytes immune to Listeria monocytogenes were about equivalent in all strains. However, clearance of Listeria by the reticuloendothelial system and early non-immune bactericidal activity of the young and old BXSB were significantly lower than in the control strains. The results indicate that the cellular-mediated immunity (CMI) of BXSB mice compared favourably with that of other strains and that there is no apparent differences between CMI of BXSB mice before the onset of disease and during the course of disease. The role of the reduced reticuloendothelial function of BXSB mice in their autoimmune disease or in their high susceptibility to infection remains to be determined.     

Biological significance of Fc receptor-bearing cells among activated T lymphocytes.

Lethally irradiated mice injected with syngeneic thymocytes and immunized with protein antigens develop specific helper T cells. If injected with semiallogeneic thymocytes, such mice generate H-2 antigen-specific cytotoxic T cells. Most spleen cells from these chimeric mice possess Fc receptors. The present results demonstrate that the development of Fc-receptor-bearing cells in thymocyte-injected irradiation chimeras seemingly is due to the physiological conditions in the mice rather than to the specific immunization. As a corollary, both helper T cells and cytotoxic T cells did not have Fc receptors, at least not in their effector state. Thus, Fc receptors on T cells would seem irrelevant to their immune function.     

Sequential interaction of macrophages, initiator T lymphocytes and recruited T lymphocytes in a cell-mediated immune response to soluble antigen.

We have investigated the sequential interaction of macrophages, initiator T lymphocytes (ITL) and recruited T lymphocytes (RTL) in the development of a T cell-mediated response to soluble antigens. Macrophages were pulsed with soluble antigens and used to sensitize ITL in vitro. The ITL were irradiated to prevent their proliferation and then injected into the footpads of syngeneic mice. Sensitized ITL were found to recruit immunospecific RTL in the draining lymph nodes, as determined by a thymidine uptake assay of the lymph node cells in vitro. The richest source of lymphocytes with ITL activity was the thymus, and progressively less activity was detectable among spleen or lymph node lymphocytes. The magnitude of the subsequent RTL response could be modified by genetic differences between the ITL and the antigen-pulsed macrophages that were used to sensitize them. Thus, ITL conveyed an immunogenic signal to RTL whose magnitude reflected the genotype of the macrophages, but whose specificity was directed by determinants of the soluble antigen.     

Expression of the Fc gamma receptor on Ly-1+ B lymphocytes

The expression of IgG Fc receptor (FcR) molecules was examined on Ly-1+ B cells and B cells tumors. FcR molecules were found on a representative Ly-1+ B cell lymphoma of the pre-B and as well as of the mature cell B phenotypes. The expression of the FcR did not change on these cells during their differentiation to B cells or to antibody-secreting cells, respectively. Ly-1+ B cells were found at low frequency (approximately 2%) in the spleens of normal mice, and in the peritoneal cavity where their representation was greater. The frequency of Ly-1+ B cells declined after birth, although their numbers increased in both organs. These Ly-1+ B cells expressed the FcR molecule throughout ontogeny. Furthermore, the amount of FcR expressed on Ly-1+ B cells was similar to the levels expressed on their "conventional" (Ly-1-) B cell counterparts. The FcR was also found on Ly-1+ B cells from autoimmune mice. The significance of the expression of FcR molecules on Ly-1+ B cells is discussed.     

C-kit+ FcR+ myelocytes are increased in cancer and prevent the proliferation of fully cytolytic T cells in the presence of immune serum

Immunogenic cancers induce both IgG antibodies and CD8(+) cytotoxic T lymphocytes (CTL). Rejection of almost all immunogenic tumors depends ultimately on CTL. When tumors grow progressively, IgG continues to be produced but CTL may no longer be demonstrable. Using syngeneic mixed lymphocyte tumor cell cultures, we found that proliferation of fully activated proliferating CTL is prevented by a small subpopulation of immature myeloid c-kit(+) FcR(+) cells, for convenience referred to as "barrier cells". Both, FcR on barrier cells and IgG linked to TGF-beta (IgG-TGF-beta) present in immune serum, are obligatory for barrier cells to prevent proliferation of CTL, suggesting that IgG-TGF-beta binds FcR to activate suppression. Growing tumors increase barrier cells in the spleen. Interfering with the cells or molecules essential for barrier cells to prevent proliferation of CTL may enhance tumor and other CD8(+) CTL-mediated immunity.