Enhancing and suppressive effects of macrophages on T-lymphocyte stimulation in vitro.

The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [¹²⁵I]UdR incorporation as a measure of lymphocyte proliferation. [¹²⁵I]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers of peritoneal macrophages beyond 5–10%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9–10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [¹²⁵I]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.     

The effects of crude and purified human gonadotropin on in vitro stimulated human lymphocyte cultures.

Crude human chorionic gonadotropin (hCG) was found to be several fold more immunosuppressive than purified hCG in human peripheral blood lymphocyte cultures stimulated by phytohemagglutinin, pokeweed, purified protein derivative and allogeneic cells in vitro. Immunosuppression by crude hCG was consistently noted at levels less than 1000 IU/ml and usually 80% inhibition was achieved with doses of 5000–10,000 IU/ml, whereas 40–50% inhibition or less was observed by purified hCG at 10,000 IU/ml. In two crude hCG preparations subjected to Sephadex G-100 chromatography, the fractions that inhibited lymphocyte cultures appeared in the eluate after the major peak of hCG activity. These data indicate that inhibitory substance(s) other than hCG are responsible for most of the immunosuppressive properties of first trimester pregnancy urine. Both crude and purified hCG were stimulatory to human lymphocytes when used alone without mitogens when cultured in fetal calf serum.     

Antibodies against cortisol block suppressive effects of corticosteroids on lymphocytes in vitro.

Lymphocyte transformation assays were used to test the ability of antibodies against cortisol to reduce bioactivity of corticosteroids in vitro. Mononuclear cells were separated from whole bovine blood and cultured in the presence of PHA alone, PHA + steroid, PHA + steroid + anticortisol, or PHA + steroid + anti-bovine serum albumin. Tritiated thymidine uptake was determined for all groups during the last 24 hr of a 72-hr culture period by scintillation counting. Polyclonal anticortisol against cortisol-bovine serum albumin conjugated in the 21 position was more effective in blocking cortisol activity than monoclonal anticortisol built against conjugates in the 3 position. The steroids that suppressed PHA-induced lymphocyte proliferation in a concentration-dependent manner were: cortisol, corticosterone, dexamethasone, prednisolone, 11-deoxycortisol, and 11-deoxycorticosterone. Aldosterone, cortisone, cholesterol, estradiol, and progesterone did not exhibit concentration-dependent effects and, thus, were not considered suppressive. These concentration-independent steroids were also the least suppressive (with the exception of aldosterone). Anticortisol was able to reduce bioactivity of suppressive corticosteroids that had an 11-hydroxy group, suggesting the antibody was primarily made against this site. Anti-BSA was not effective in blocking corticosteroid activity, but it did enhance proliferation of lymphocytes if added in combination with weakly suppressive steroids. Anticortisol also had an enhancing effect when added with some weakly suppressive steroids. We conclude that antibodies against cortisol are capable of reducing bioactivity of steroids that strongly suppress lymphocyte proliferation. Additionally, the 11-hydroxy group may be an important antigenic determinant of steroid molecules.     

Antibody responses of human lymphocytes in vitro. Enhancing effects of hydrocortisone.

Cultures of human tonsillar lymphocytes stimulated by pokeweed mitogen in the presence of hydrocortisone succinate generate more sheep red blood cell-specific plaque-forming cells than when the hydrocortisone is absent. Such enhancement is even more marked when sheep red blood cells and pokeweed mitogen are present in cultures. This contrasts with the inability of sheep red blood cells to enhance pokeweed mitogen-stimulated response in cultures lacking hydrocortisone. Hydrocortisone causes greater enhancement when the concentration of cells in culture is high than when it is low. These data are consistent with hydrocortisone inhibiting the activity of suppressor cells which would otherwise mask helper effects.     

Kinetics of human lymphocyte responses in vitro: the phase of exponential growth

The kinetics of thymidine uptake in human peripheral lymphocytes stimulated by allogenic cells, antigen E (ragweed allergen) and a variety of mitogens can generally be divided into four consecutive phases. First, a lag period with no increase in thymidine uptake, then a short period of rapid change in uptake, followed by a log-linear growth period and finally a decay phase. In this report we examine in detail the characteristics of the third, log-linear growth phase. Since, as discussed in the preceding paper, thymidine uptake is proportional to the number of cells acumulating thymidine, we can calculate from the log-linear growth period an apparent doubling time. We show that for five different stimulating agents the cells reach a log-linear growth phase of varying length and that the doubling times show little variation. This invariance indicates that, despite possible variation in cell death and recruitment rates, the rate of proliferation is in all cases dominated by the generation time of human lymphocytes.     

Inability of gestational hormones to account for the inhibitory effects of pregnancy plasmas on lymphocyte responses in vitro.

Plasma from pregnant women has a marked inhibitory effect on lymphocyte responses in vitro. While much evidence suggests that this is due to an immunologic mechanism, an apparent lack of specificity and the known suppressive effects of several hormones on immune function has led to speculation that the inhibitory effects could be due to increased concentrations of gestational hormones. We have investigated the effects of a wide range of concentrations of estrone, estradiol, estriol, progesterone, human chorionic gonadotropin (HCG), and hydrocortisone on lymphocyte responses to mitogens and allogeneic cells. None of these hormones were capable of inhibiting lymphocyte DNA synthesis even at concentrations several times the maximum physiologic plasma levels occurring during pregnancy. Very high, supraphysiologic concentrations were found to be inhibitory. In investigating the mechanism of the hormonal inhibition we found that if they were removed from the media at various times after initiation of culture, the estradiol, HCG, and to a lesser extent the hydrocortisone effects were all reversible. Estradiol and HCG differed from hydrocortisone in that the former were inhibitory only when added at the initiation of culture, whereas hydrocortisone was inhibitory even when added 24 hr later. In summary, while extremely high concentrations of gestational hormones are inhibitory, the quantities which occur physiologically in gestational plasmas are not able to suppress lymphocyte responses and thus cannot account for their inhibitory effects.     

Immune responses to isolated human cytomegalovirus envelope proteins.

A group of envelope proteins of human cytomegalovirus, gA protein (L. Pereira, M. Hoffman, M. Tatsuno, and D. Dondero, Virology 139:73-86, 1984; L. Pereira, p. 383-404, in B. Roizman, ed., The herpesviruses, vol. 3, 1985), and two protein mixtures (58,000-molecular-weight [58K]-66K and 130K-66K), separated by serial columns prepared with anti-gA immunoglobulin G from sera of immunized guinea pigs, induced neutralizing antibodies and a cellular immune response in the animals. The gA is a disulfide-linked protein complex consisting of high-molecular-weight (greater than 200K), 130K-150K, and 55K-58K proteins.     

Histamine suppression of human lymphocyte responses to mitogens.

Exogenously added histamine in non-cytotoxic concentrations (10^?5?10^?3M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as $\text{1}\text{2}$ hr in the beginning of the culture. Histamine, in concentrations as high as 10^?3M, did not cause increased release of isotope from ⁵¹Cr-labeled lymphocytes following 4 hr of incubation. The histamine H₂ receptor antagonist, metiamide, but not the H₁ receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.     

Cytotoxic and proliferative lymphocyte responses to allogeneic and xenogeneic antigens in vitro.

In vitro lymphoproliferative responses to foreign histocompatibility antigens are phylogenetically restricted. Responses occur most readily to allogeneic or closely related xenogeneic leucocytes, but not to unrelated xenogeneic cells. Specific cytotoxic T cell responses to foreign histocompatibility antigens show the same phylogenetic restriction. This lack of xenoreactivity is not due to a lack of precursor cells for the xenoantigens; guinea-pig lymphocytes, although normally unresponsive to mouse antigens, have a similar precursor frequency for these antigens as do lymphocytes of allogeneic mouse strains. Specific cytotoxic responses of guinea-pig lymphocytes to mouse antigens can be generated if a factor released from con A stimulated guinea-pig spleen cells is added to the culture medium. The factor produced by con A-activated spleen cells (CS) is also phylogenetically restricted in its action; CS must be obtained from animals homologous with the donor of the responding lymphocytes.     

In vitro murine lymphocyte blastogenic responses to Cryptosporidium parvum.

Spleen and mesenteric lymph node lymphocytes from both Cryptosporidium parvum-exposed and unexposed mice were cultured with antigen (Ag) prepared from C. parvum oocysts. Spleen lymphocytes from oral-, intraperitoneal-, or oral + intraperitoneal-exposed mice did not respond significantly (P greater than 0.05) to Ag stimulation. Spleen lymphocytes from multioral-exposed mice, however, demonstrated significant (P less than or equal to 0.01) Ag-specific blastogenesis. Mesenteric lymph node lymphocytes did not respond to in vitro Ag stimulation regardless of the route of in vivo priming. These results demonstrate an in vitro cell-mediated immune response against C. parvum by lymphocytes in murine spleen.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.     

Effect of murine cytomegalovirus on the in vitro responses of T and B cells to mitogens.

Both in vitro and in vivo murine cytomegalovirus (MCMV) infection depressed the responses of lymphocytes to both B and T cell mitogens. The possibilities that macrophages or nonspecific T cell inhibition of B cells might account for the depressed responses were eliminated. In vitro data suggested that B cell responses are more susceptible to this depression than T cell responses. The possibility that the depression of T cell responses is not a direct effect of viral infection of lymphocytes is discussed. To investigate further the interaction between B and T lymphocytes and MCMV, mice with B and T cell deficiences were studied. A comparison of the susceptibility of athymic Nu/Nu mice and T cell competent Nu/+ littermates to MCMV showed that the LD50 for Nu/Nu mice is 10-fold lower than that for Nu/+ mice, but Nu/+ mice given an LD50 of virus died much sooner after infection than Nu/Nu mice given an LD50. Pathogenic mechanisms responsible for death may be different in these two groups of mice. Similarly the MCMV LD50 for B cell-deficient mice (treated with goat anti-mouse IgM serum) was 10-fold lower than the LD50 for mice treated with normal goat serum, but given an LD50 of virus, the latter died sooner after infection than the former. In contrast, there was little difference between the LD50 or time of death after MCMV infection of CBA x DBA F1 male mice (which are deficient in their response to thymic independent antigens) and their normal littermates, the CBA x DBA F1 female mice.     

Human immune responses to major human cytomegalovirus glycoprotein complexes.

Sera from both human cytomegalovirus (HCMV)-seropositive adults and infants with congenital HCMV infection recognized two major HCMV glycoprotein complexes. However, proliferative responses of peripheral blood mononuclear cells to these complexes varied among seropositive adults and were not detected in any of the infants. Thus, these glycoproteins alone may not be sufficient to develop a subviral HCMV vaccine.     

Regulation of human cytolytic lymphocyte responses by interleukin-12.

IL-12 is a heterodimeric cytokine which has been shown to cause the proliferation of activated T and NK cells, to enhance the lytic activity of NK cells, and to induce IFN-gamma production by resting and activated T and NK cells. We previously reported that IL-12 could synergize with IL-2 to activate human LAK cells in the presence of hydrocortisone but that IL-12 alone was inactive. We herein show that in the absence of hydrocortisone, IL-12 by itself can activate human LAK cells. IL-12-induced LAK cell activity was mediated predominantly by CD56+ lymphocytes. Activation of LAK cells by IL-12 appeared to be independent of IL-2 since it was not inhibited by neutralizing anti-human IL-2. However, IL-12- and IL-2-induced LAK cell activation could be partially inhibited by anti-human TNF-alpha. Moreover, IL-12 produced in situ appeared to play a role in IL-2-induced LAK cell activation since rat monoclonal antibodies to human IL-12 could partially inhibit the generation of LAK cells in response to IL-2. In addition to its effects on LAK cell responses, IL-12 could facilitate specific allogeneic human CTL responses. However, IL-12-facilitated CTL responses were blocked by neutralizing anti-human IL-2 indicating a requirement for IL-2 produced in situ. The ability of IL-12 to facilitate both nonspecific LAK and specific CTL responses suggests that it may be useful as a therapeutic agent against some tumors and infectious diseases.     

Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells

The ability of two different human professional APCs, specifically macrophages (Mphi) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mphi and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mphi, were able to prime a CTL response against the B14-restricted p24gag 298-306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259-267 epitope. In contrast, Mphi were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mphi and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mphi in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes.