Human suppressor factors constitutively produced by T-T cell hybridomas: functional and biochemical characterization

We have recently developed a new method (Hybridoma 6:589, 1987) for the generation of human T-T cell hybrids. This method is based on a new selection procedure that involves cloning the hybrids in soft agar, screening by HLA-typing or appropriate functional tests and recloning by limiting dilution. T-T cell hybrids were separated from the parent line on the basis of their ability to form colonies in soft agar, whereas the parent lymphoblastoid T cell lines did not. HAT medium was not used in our selection procedure. Using this method, we have succeeded in developing human T-T cell hybrids (as determined by HLA-typing) constitutively producing B cell growth factor (BCGF) (Hybridoma 6:589, 1987) or suppressor factors. These hybrids were obtained by fusing MLC or Con A T cell blasts with cells from the Molt 4 or Jurkat lymphoblastoid T cell lines. T-T cell hybridomas, derived by fusing Con A-stimulated lymphocytes with cells from the Jurkat T cell line, produced suppressor factors inhibiting: (1) proliferative response in vitro of human peripheral blood mononuclear leukocytes to mitogens and to allogeneic cells in mixed lymphocyte culture; and (2) immunoglobulin synthesis and secretion by mononuclear leukocytes in the PWM-induced differentiation system in vitro. A suppressor factor with these inhibitory properties was also identified in supernatants of the Jurkat T cell line. These suppressor factors were ammonium sulphate precipitable, pH 2 labile, non-dialyzable and they were inactivated by treatment at 56 degrees C for 30 minutes. They exhibited a molecular weight in the range of 50,000-70,000, as determined by gel filtration, and were not gamma or alpha interferon or lymphotoxin/TNF. They did not lyse human lymphoblastoid tumor cell lines nor did they affect the viability and cell numbers of human mononuclear cells even after prolonged incubation (88 hr). They appeared to be cytostatic rather than cytotoxic molecules. The Jurkat suppressor factor is different from those produced by the hybrids on the basis of: (a) different isoelectric points; and (b) the ability of the Jurkat factor to arrest proliferation to PHA of human mononuclear cells in the S phase, whereas the 160 and 169 factors arrest proliferation at the G1 phase of the cell cycle. Certain of these suppressor factors (produced by the hybrids 153, 160, 170, and the Jurkat T cell line) also inhibited proliferative responses of mouse lymphocytes in vitro. In contrast, suppressor factors produced by the 169 and 77 hybrids did not inhibit any murine responses.     

Role and function of antigen nonspecific suppressor factors

Although antigen-nonspecific suppressor factors described by various investigators appear to exhibit a certain amount of heterogeneity in both physical and biological properties, these proteins also exhibit significant similarities. Nonspecific suppressor factors are generally produced by Ly 2+ (murine) or OKT8+ (human) T lymphocytes. One protein, soluble immune response suppressor (SIRS), is produced by T lymphocytes after incubation with mitogens, interferons, or histamine, and must be activated by peroxides to inhibit cell division or immune function. SIRS appears to inhibit cell division by causing oxidation of a portion of cellular protein sylfhydryls and, in particular, causes a decrease in intracellular levels of deoxyribonucleotide triphosphates. This decrease is readily reversed by sulhydryl reducing agents, such as 2-mercaptoethanol. The activity of SIRS and other suppressor factors is inhibited by growth factors, such as interleukin 2, and the activity of interleukin 2 is inhibited by antigen-nonspecific suppressor factors. Further, SIRS or SIRS-like proteins are produced during various diseases associated with suppressed immune responsiveness including acquired immune deficiency syndrome, schistosomiasis, and nephrotic syndrome. These data suggest that antigen-nonspecific suppressor factors may have an important physiological role in regulating immune responses and cell division in general.     

Studies on the mechanism of peptidoglycan- and lipopolysaccharide-induced polyclonal activation.

Peptidoglycan (PG) and lipopolysaccharide (LPS) are T cell-independent B cell mitogens and polyclonal activators in mice. The mechanism of in vitro proliferation and polyclonal activation of mouse splenocytes induced by PG from Staphylococcus aureus and LPS from Escherichia coli was further studied by using [3H]thymidine incorporation and protein A hemolytic plaque assays. Concanavalin A-generated suppressor cells suppressed both polyclonal and proliferative responses induced by PG, LPS, and pokeweed mitogen. The suppression of the proliferative responses was similar for all these mitogens, but was significantly less pronounced than the suppression of the polyclonal antibody response. Polyclonal activation induced by LPS was the most susceptible to suppression by concanavalin A-generated suppressor cells, and the suppression was significantly greater than in the PG-induced polyclonal response. Also, PG-induced polyclonal activation was not susceptible to inhibition by polymyxin B, which is an inhibitor of other B cell mitogens and polyclonal activators. For optimal generation of immunoglobulin-secreting cells, PG of LPS had to be present for at least 48 h after the initiation of the cultures. Removal of the mitogens after 4 or 24 h of incubation resulted in a suboptimal response. For effective induction of the proliferative response, the mitogens had to be present in cultures for over 24 h. Polyclonal-activating properties of staphylococcal cell wall components were also compared. PG was by far the most potent inducer of polyclonal antibodies. Teichoic acid was not active as a polyclonal activator, whereas purified cell wall and protein A were very weak inducers of polyclonal antibodies. These studies demonstrate that PG, in addition to LPS, can be a useful probe for studies on polyclonal activation.     

Cyclosporin A has differential effects on the responses of murine B cells to TI antigens and B-cell mitogens.

The effect of cyclosporin A (CyA) on the response of murine splenocytes to B-cell mitogens, TI-1 and TI-2 antigens was investigated. The proliferative response to LPS was found to be four- to five-fold less sensitive to inhibition than that to dextran sulphate. Antibody responses to a TI-2 antigen in vivo were suppressed by CyA treatment, whereas responses to the TI-1 antigen DNP-LPS were markedly enhanced. Enhanced antibody responses to DNP-LPS were also demonstrable in vitro in the presence of CyA, and the enhancement was not removed by T-cell depletion. LPS-induced antibody production in vitro was enhanced at the same CyA concentrations that inhibited proliferation by 40-50%. The implications of these findings for the mechanism of action of CyA and for our understanding of B-cell differentiation are discussed.     

A new method for the development of human T-T cell hybrids without the use of HAT medium

We report here a new method for the development of human T-T cell hybrids by fusing mitogen- or alloantigen-stimulated T cells with non-mutagenized cells from human lymphoblastoid T cell lines. This method is based on a new selection procedure where the hybrids are separated from the parent T cell line on the basis of their ability to form colonies in soft agar. In contrast, cells from lymphoblastoid T cell lines Molt-4 and Jurkat do not form colonies in agar. Hybridoma colonies are retrieved from the agar plates, expanded in culture, screened by HLA-typing and appropriate functional tests and recloned several times by limiting dilution. HAT medium, which contains thymidine that appears to be toxic to the hybrids, is not used in our selection procedure. Using this method, we developed human T-T cell hybridomas (as determined by HLA-typing) producing B-cell growth factor (BCGF) either constitutively or after induction with Concanavalin A (Con A). Certain other T-T cell hybrids produced suppressor factor, significantly inhibiting proliferative responses of human peripheral blood mononuclear leukocytes to PHA, Con A and allogeneic cells in mixed lymphocyte culture.     

Immunostimulatory Actions of Lactobacilli: Mitogenic Induction of Antibody Production and Spleen Cell Proliferation by Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus

We investigated the ability of heat-treated Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus to act as direct inducers of antibody production and cellular proliferation. Yogurt starter-derived strains of L. delbrueckii subsp. bulgaricus and L. acidophilus induced high levels of antibody production by murine splenocytes in vitro . Both IgM and IgG isotypes were produced. In contrast, two different strains, L. delbrueckii subsp. bulgaricus (ATCC 11977) and L. acidophilus (ATCC 521) did not induce antibody production by murine splenocytes. While all four strains were able to induce some degree of splenocyte proliferation, the yogurt starter-derived strain of L. acidophilus was the most potent inducer. These results indicate a mitogenic effect of certain strains of lactobacilli on murine splenocytes, resulting in polyclonal antibody production. The ability of heat-treated lactic acid bacteria to induce antibody production and proliferation suggests the involvement of a heat-resistant structural component in non-specific activation of the immune system by these strains of lactic acid bacteria.     

Amotosalen-treated donor T cells have polyclonal antigen-specific long-term function without graft-versus-host disease after allogeneic bone marrow transplantation.

We have previously shown that amotosalen HCl (S-59 psoralen)-treated donor splenocytes, which have limited proliferative capacity in vitro, can protect major histocompatibility complex-mismatched bone marrow transplant (BMT) recipients from lethal murine cytomegalovirus infection without causing graft-versus-host disease. In this study, we further investigated the effects of amotosalen-treated donor T cells on immune reconstitution after allogeneic BMT. We were surprised to find that amotosalen-treated donor T cells persisted long-term in vivo, comprising 6% to 10% on average of the T-cell compartment of transplant recipients at 4 months after transplantation. Donor T cells derived from amotosalen-treated splenocytes were predominantly polyclonal CD44 hi/int CD8 + memory T cells and were functionally active, synthesizing interferon gamma in response to stimulation with murine cytomegalovirus antigen. Amotosalen-treated donor T cells, reisolated from BMT recipients' spleens >/=4 months after transplantation, proliferated in vitro, thus indicating repair of amotosalen-mediated DNA cross-links. Compared with infusion of untreated donor splenocytes, amotosalen-treated cells enhanced thymopoiesis by bone marrow-derived stem cells in BMT recipients. However, amotosalen treatment abrogated the thymopoietic activity of lymphoid progenitor cells among the donor splenocytes. Thus, infusion of amotosalen-treated donor T cells produced rapid immune reconstitution after major histocompatibility complex-mismatched BMT by transferring long-lived polyclonal memory T cells with antiviral activity and also by enhancing bone marrow-derived thymopoiesis. This is a novel approach to adoptive immunotherapy in allogeneic BMT.     

Differential effects of human alpha and gamma interferon on mixed lymphocyte culture and on T-cell-mediated cytotoxicity. Inhibition of proliferation but not of IL-2 production by alpha interferons

We compared the effects of natural and recombinant (r) alpha (IFN-alpha) and gamma (IFN-gamma) interferons on the proliferative responses of human peripheral blood mononuclear cells to mitogens and allogeneic cells in mixed lymphocyte culture (MLC) and on the generation of specific T-cell-mediated cytotoxicity. In 14 of 19 donors, natural IFN-gamma and rIFN-gamma had no significant effect on the proliferative responses to mitogens or allogeneic cells in MLC, even at very high IFN-gamma concentrations (10,000 U/ml). In the remaining 5 donors, a statistically significant (p less than 0.001) enhancement by 49 +/- 8% of the proliferative responses was observed. In contrast, natural IFN-alpha and rIFN-alpha 2 significantly inhibited (p less than 0.001) proliferative responses to mitogens and to allogeneic cells, even at concentrations as low as 10 U/ml, in agreement with previous reports. Although natural and recombinant IFN-alpha significantly inhibited these proliferative responses, they did not affect interleukin-2 (IL-2) production in these cultures, suggesting that they inhibit proliferation by a mechanism that does not involve inhibition of IL-2 production. rIFN-gamma did not affect the generation of specific cytotoxicity in MLC, although it was significantly enhanced by natural IFN-alpha and rIFN-alpha 2. Additionally, we compared the ability of human rIFN-alpha subtypes to inhibit proliferative responses to allogeneic cells in MLC. rIFN-alpha 2, rIFN-alpha 4, and rIFN alpha 7 displayed the most potent inhibitory activity of allogeneic responses and were active at concentrations as low as 0.3-0.6 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunosuppression induced by nitric oxide and its inhibition by interleukin-4.

Mice immunized with attenuated Salmonella typhimurium, strain SL3235, while protected against virulent challenge, are unable to mount in vivo and in vitro antibody responses to non-Salmonella antigens, such as tetanus toxoid and sheep red blood cells, and exhibit profoundly suppressed responses to B and T cell mitogens. Suppression of antibody responses is mediated by macrophage (M phi)-released soluble factors, and is completely reversed by treatment with interleukin (IL)-4. The present report identifies the suppressor factor as nitric oxide (NO), and provides evidence for a mechanism by which IL-4 abrogates suppression. Suppressed antibody responses correlated with high levels of NO secretion by splenocytes of SL3235-immunized mice. NO production was observed only in cultures consisting of the adherent cell fraction of immune splenocytes. Further, immunosuppression was reversed by NG-monomethyl-L-arginine (NMLA), a competitive inhibitor of NO synthesis, and was completely blocked by the addition of excess L-arginine. Treatment with IL-4, or anti-interferon (IFN)-gamma monoclonal antibody (mAb), also abrogated suppression. Optimal reversal of suppression was observed only when NMLA, IL-4, or anti-IFN-gamma mAb, was added at day 0 of the 5-day plaque-forming cell assay. Treatment with either IL-4 or anti-IFN-gamma mAb also lead to a sharp inhibition of NO production by immune spleen cells. Moreover, the addition of IL-4 to splenic adherent M phi inhibited their ability to generate NO. Our data characterize an immunoregulatory pathway, involving IFN-gamma and NO, by which M phi mediate immunosuppression and identify IL-4 as a potent inhibitor of this pathway.     

The immunological profile of a new immunomodulatory agent, oxamisole

Oxamisole is a T-cell immunorestorative agent when administered by the oral (p.o.) route. It has little or no effect on the IgM or IgG responses to the T-dependent antigen, sheep erythrocytes (SRBC), in normal mice but augments antibody production in immunodeficient animals. Unlike the response to SRBC, the humoral immunocompetence of both normal and immunosuppressed animals sensitized with the T-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS) was unaffected by oxamisole. Oxamisole restored cellular immunocompetence, as evidenced by an increase in the in vitro proliferative response of normal murine splenocytes to T-cell mitogens, while decreasing B-cell mitogenic responses. This indicates that oxamisole may selectively restore T-cell function. However, oxamisole did not significantly modify the classical T-cell-mediated delayed hypersensitivity responses to either the protein antigen methylated bovine serum albumin or to the contact-sensitizing antigen oxazolone. When assayed in vitro, oxamisole enhanced macrophage chemotactic function but not phagocytic function, suggesting a potential stimulation of the reticuloendothelial system. In vivo studies failed to demonstrate any consistent significant activation of murine macrophage function following oral dosing with oxamisole.     

Specific suppression of antibody responses in vivo. I. Effects of in vitro produced suppressor factor.

KLH-specific suppressor factors produced in vitro efficiently diminished primary and secondary responses to trinitrophenyl keyhole limpet haemocyanin (TNP-KLH) in vivo. Both IgM and IgG responses were approximately equally affected, These suppressor factors were not genetically restricted, as allogeneic suppressor factors worked as efficiently as syngeneic factors. Furthermore, xenogeneic human suppressor factors were effective in mice and suppressed the responses as efficiently as syngeneic factors. The kinetics of the response in suppressed and non-suppressed mice was the same, indicating that the magnitude of the response was affected and not merely its time course. Prior injection with suppressor factor did not cause suppression of response, while suppressor factor injected at the same time as or soon after the antigen did, suggesting that it might act at the effector stage. The mechanism of action of this unrestricted suppressor factor, and the use of mouse model for in vivo testing of human suppressor factors is discussed.     

Induction of a T-cell mediated suppressor activity by soluble products from antigen-specific helper/inducer human T-cell lines.

Influenza virus-specific (A/X31) long-term cultured human T-cell lines belonging to the helper/inducer T-cell set, produce high potency antigen specific helper factors which induce in vitro antibody production to A/X31 by autologous B cells, as well as small and variable amounts of non-specific helper factors. When added to unseparated peripheral blood mononuclear cells, both cultured T cells and their supernatants suppress in vitro antibody synthesis as measured by a solid phase enzyme-linked immunoassay, and T-cell proliferation to antigens and allogeneic cells, but not to mitogens. This phenomenon was further analysed and could be separated into several steps: (i) the production of suppressor inducer factor(s) by the T-cell lines which are distinct from the helper molecules; (ii) activation of T cells belonging to the suppressor/cytotoxic subset as defined by monoclonal antibodies, a process which is antigen-independent and non-genetically restricted, and is optimal with 18 hr incubation; (iii) the activated T cells non-specific suppress antibody production and antigen-induced or allogeneic cell-induced T-cell proliferation. Thus, antigen-specifically activated T-inducer cells exert multiple activities, including specific and non-specific help and non-specific induction of T suppressor cells.     

Ubiquitin-like polypeptide inhibits the proliferative response of T cells in vivo

The monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic Ag-nonspecific suppressive functions. Recently, we demonstrated that the recombinant form of the ubiquitin-like segment (rUbi-L) of MNSFbeta, a 15.6 kDa-protein consisting of a polypeptide with 36% homology with ubiquitin fused to the ribosomal protein S30, presented an antigen-nonspecific immunoregulatory action in a manner similar to native MNSF. Although this cytokine has been characterized in vitro, little is known about its effects in vivo. Thus, we investigated whether rUbi-L shows a suppressor activity in vivo. The proliferative response of Con A (5 microg/ml)-stimulated splenocytes of mice treated with rUbi-L (500 ng/body) was notably decreased in a dose-dependent manner (max. 57+/-20%). In contrast, administration of high dose ubiquitin (50 microg/body) showed a little, but significant, effect (30+/-7%). Interestingly, concomitant addition of ubiquitin inhibited Ubi-L-induced suppression. Mice injected with rUbi-L without gelatin did not show any suppressive effect. NA4 (1microg/body), a neutralizing monoclonal antibody against rUbi-L, abolished the Ubi-L-mediated suppression. Therefore, ubiquitin-like polypeptide may be implicated in the immune responses in vivo.     

Effect of lipid A-associated protein and lipid A on the expression of lipopolysaccharide activity. I. Immunological activity.

A detailed investigation has been made of the contribution of the various chemical moieties of bacterial endotoxins, namely lipid A-associated protein (LAP), lipid A and O-antigen polysaccharide to a number of the immunological activities of these active bacterial products. Advantage was taken of the availability of antigenically identical endotoxin preparations from Escherichia coli 0111:B4 which differed greatly in their content of LAP and/or lipid A. The capacity to initiate in vitro proliferative responses in murine splenocytes was in a large part related to the presence of LAP with a less potent, although still critical, dependence upon lipid A. On the other hand, the in vivo polyclonal antibody response was dependent only upon lipid A. In this respect, the presence of LAP had no apparent effect on the stimulation of nonspecific low affinity antibody. All preparations, regardless of LAP and lipid A content, stimulated similar in vivo enhancement of antibody responses to a protein antigen (adjuvanticity) and specific immune responses to the endotoxin polysaccharide antigen. The results emphasize the lack of correlation between in vitro B lymphocyte proliferative responses and in vivo immunostimulatory responses of bacterial endotoxin preparations. These data also suggest a minimal contribution of LAP to in vivo responses and an extremely limited contribution of lipid A to the adjuvant activity and the primary immune response to O-antigen polysaccharide.     

A modification of the in vivo mixed lymphocyte reaction and rapamycin's effect in this model.

Rapamycin, a novel macrocyclic immunosuppressive agent, suppresses murine T cell activation in vitro by mechanisms distinct from cyclosporin A (CsA). This study was designed to examine rapamycin and CsA in the host vs graft popliteal lymph node (PLN) model, an in vivo system of T cell-dependent lymphocyte activation. The PLN procedure was modified by using irradiated CTLL-2 cells of C57BL/6 origin, instead of primary mouse splenocytes, as the allogeneic stimulus in C3H/HeN recipient mice. PLN cell proliferation was determined by [3H]-thymidine uptake. We found that the host lymphocyte proliferative response to CTLL-2 cells (H-2b) is greater than the response to mouse Balb/c splenocytes (H-2d). Rapamycin (ip or po) produced a dose-related inhibition of the in vivo mixed lymphocyte reaction. By contrast, the effects of CsA and FK-506 were not dose related within the same dose range (0.006-12 mg/kg). These data indicate that rapamycin is an effective immunosuppressive agent and confirm its ability to affect the allogeneic T cell response in vivo. Furthermore, the pharmacological data suggest that this PLN model utilizing irradiated CTLL-2 cells as an allogeneic stimulus provides a reproducible system to examine mixed lymphocyte reactions in vivo.     

Nitric oxide synthesis is depressed in Bos indicus cattle infected with Trypanosoma congolense and Trypanosoma vivax and does not mediate T-cell suppression.

Infection with African trypanosomes causes the diseases sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. Suppression of cellular immune responses is a feature of trypanosomiasis in bovine, human, and murine hosts. Some aspects of immunosuppression in the murine model are mediated by nitric oxide (NO) produced by gamma interferon (IFN-gamma)-activated macrophages. We have investigated whether a similar mechanism is responsible for T-cell unresponsiveness in bovine trypanosomiasis. Bovine monocytes and macrophages from uninfected cattle and activated in vitro with IFN-gamma produced NO; however, this response was down-regulated in infected cattle. Similarly, the expression of inducible NO synthase messenger RNA was depressed in macrophages of infected cattle. Proliferation of mononuclear cells of trypanosome-infected cattle cultured with mitogen or trypanosome antigens was unchanged by the addition of an NO synthase inhibitor. Lymphocytes of infected cattle secreted interleukins with T-cell growth factor activity after in vitro activation with mitogens but not after activation with trypanosome antigens. Although lymph node cells secreted IFN-gamma after in vitro activation, ex vivo expression of mRNA was depressed. In contrast, the level of expression of interleukin 10 mRNA was higher during infection. We conclude that NO is not involved in the loss of T-cell proliferative function associated with trypanosomiasis in cattle and that, in contrast to the mouse model, the capacity of monocytes and macrophages to produce NO is actually down-regulated in infected cattle.     

A novel cytotoxicity assay to evaluate antigen-specific CTL responses using a colorimetric substrate for Granzyme B

We have utilized the unique enzymatic properties of a key cytotoxic mediator in target cell destruction, Granzyme B (GrB), to establish an attractive alternative to 51Cr-release assays for the assessment of antigen-specific CTL responses. A number of potential colorimetric peptide substrates were compared to evaluate levels of GrB activity in cytolytic cells. The most specific and sensitive substrate for GrB was Ac-IEPD-pNA, as shown by the minimal enzymatic hydrolysis in apoptotic Jurkat cells and strong hydrolysis in human NK cells. When human peripheral blood lymphocytes were stimulated in vitro, elevated GrB levels were detected by both Ac-IEPD-pNA and a GrB ELISA. Analysis of allo-antigen-specific murine CTLs revealed that GrB exocytosis was only detectable upon challenge with appropriate allogeneic target cells and strongly correlated to 51Cr-release data. The validity of using Ac-IEPD-pNA in vaccine trials was demonstrated in mice immunized with allogeneic P815 cells, where GrB enzymatic activity was measurable in ex vivo splenocytes cell cultures only upon co-incubation with P815 targets. Additionally, influenza-infected mice were also assessed for GrB activity following in vitro peptide-stimulation of splenocytes and strongly reflected both peptide-specific tetramer staining and 51Cr-release results. The novel cytotoxic assay presented here should give investigators a sensitive, cross-species, nonradioactive alternative to 51Cr-release assays as a means to assess antigen-specific CTL responses in vaccine trials.     

Cellular Immunity in Human Milk

ABSTRACT: The responses of human milk lymphocytes (MIL) to a variety of immunogenic stimuli were studied and compared to those of peripheral blood lymphocytes (PBL) from the milk donors. MIL showed a decreased proliferative response to mitogens and allogeneic leukocytes in vitro but displayed the ability to stimulate alloreactivity equivalent to PBL. Neither pretreatment with cell-free autologous milk nor co-cultured MIL were capable of suppressing the proliferative responses of PBL. Moreover, macrophages isolated from milk and pulsed with soluble antigen or allogeneic cells effectively induced proliferation by peripheral blood T cells whereas the response of milk nonadherent cells to antigen presented by peripheral macrophages was very low. MIL respond better to pathogenic enteric E. coli than PBL but not as well as PBL to Yersinia enterocolitica. Treatment of MIL with monoclonal antibodies cytotoxic for T cells abolished their response to bacterial antigens. Application of an anti HLA class II antigen monoclonal antibody to mixed lymphocyte or lymphocyte-bacteria cultures resulted in substantial inhibition of the MIL response similarly to that of PBL. The relevance of these data to the immunological needs of the neonate are discussed.     

In vivo depletion of BoT4 (CD4) and of non-T4/T8 lymphocyte subsets in cattle with monoclonal antibodies

The effect on certain immune responses of depleting two distinct lymphocyte subpopulations in vivo by inoculating calves with monoclonal antibodies (mAb) was examined. An mAb directed against the BoT4 antigen (the bovine homologue of CD4) effectively removed the BoT4+ lymphocytes from peripheral blood mononuclear cells (PBM). Compared to controls, treated calves showed a reduced antibody response to human O red blood cells and to ovalbumin. PBM prepared from BoT4-depleted animals also had a significantly reduced ability to respond in vitro to the mitogens phytohemagglutinin, concanavalin A and pokeweed mitogen. An mAb directed against a second numerically large bovine lymphocyte subpopulation i.e. BoT2-, BoT4-, BoT8- (CD2-, CD4-, CD8-), that may be homologous to the CD4-, CD8- cells in man and rodents that synthesize the gamma/delta+ T cell receptor, was also used for in vivo depletion. Compared to controls, calves depleted of this subpopulation showed an enhanced antibody response. The proliferative response of PBM to pokeweed mitogen was also significantly increased but responses to concanavalin A and phytohemagglutinin remained unchanged. The results suggest this lymphocyte subpopulation has a nonspecific suppressor activity acting on B cell responses either directly or through an effect on T helper cells. The non-T4/T8 cells are found extensively in the epithelium and lamina propria of the mucosa of the alimentary tract but not in T cell areas of the lymph nodes, tonsil and spleen. These non-T4/T8 cells may thus be, or contain, an intraepithelial lymphocyte population with a suppressor function.