Immunorestoration of old mice by injection of thymus extract: Enhancement of T cell-T cell cooperation in the in vitro antibody response

The effect of injecting a thymus extract (TP-1 or Thy-5) into immunodeficient old mice on the in vitro antibody response of their spleen cells was investigated by techniques suitable for dissecting out T- and B-cell reactivities. The anti-TNP antibody response of HRBC-primed spleen cells from old mice uninjected or injected with TP-1 or Thy-5 was elicited in vitro by TNP-HRBC or TNP-Ficoll. Treatment with TP-1 or Thy-5 was found to induce only a slight increase in the anti-TNP antibody response to both immunogens. The helper activity of HRBC-primed spleen cells from untreated or treated old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from young mice and the immunogen TNP-HRBC. Under these conditions it was found that both thymus extracts are very effective in restoring T cell-T cell cooperation in the generation of helper cell activity.     

Age restriction in antigen-specific immunosuppression

Age-related alterations of antigen-specific T cell-mediated suppression have been examined in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. Inducer suppressor T cells (Tsi) were activated in mice at the age of 3 mo (young) or 18 mo (old) by i.v. injection of NP-conjugated syngeneic spleen cells (SC). Spleen cells from the NP-SC-injected mice were subcultured in vitro with spleen cells from normal young or old mice to generate transducer suppressor T cells (Tst). Four days later subcultured cells were added to responder cell cultures 1 day before the PFC assays to trigger effector suppressor T cells (Tse). Responder cell cultures, containing NP-conjugated horse red blood cells (HRBC) and spleen cells from HRBC-primed young or old mice, were assayed on day 4 for anti-NP and anti-HRBC PFC. Suppression was found to be antigen specific and age restricted. NP-specific suppressor cells are easily induced in subculture if the Tsi and Tst cell populations are both derived from young or old mice. Conversely, if Tsi cells from young or old mice are subcultured with Tst cells from mice of a different age, suppression of the anti-NP PFC response is hardly observed. Age restriction was also found to operate in the interactions between subcultured and responder cell populations, indicating that age-matching is required for effective triggering of Tse cells by Tst cells. These results altogether suggest that aging may affect the recognition repertoire expressed in suppressor T cell subsets. Moreover, the finding that suppression is less efficient when exerted on responder spleen cells from old than from young mice provides an explanation for the increased frequency of autoimmune disorders in aging.     

Enhancement of suppressor T cell activity by injection of anti-IFN-gamma monoclonal antibody

In the present study, the contribution of IFN-gamma to the generation of helper activity in mice was investigated by use of anti-mouse IFN-gamma rat mAB (AN 18.17.24). This mAb was alum precipitated and injected i.p. before or after carrier priming. Results show that spleen cell helper activity is markedly inhibited by anti-IFN-gamma mAb injection. This inhibition is time and dose dependent, and counteracted by IFN-gamma administration. Thus, the anti-IFN-gamma mAb appears to inhibit helper cell activity by neutralization of the IFN-gamma required for the antibody response. Moreover, AN 18.17.24 mAb injection results in increased activation of Lyt-2+ T cells which markedly suppress Th activity. These findings altogether indicate that besides the activation of macrophages and Th, IFN-gamma seems to exert a negative interference in suppressor T lymphocyte circuits and, as a consequence, to inhibit immunosuppression.     

Defective helper T-cell activity in LPS-unresponsive C3H/HeJ mice

C3H/HeJ mice, unresponsive to LPS, exhibit a defective ability to mount antibody responses to T-dependent immunogens. The anti-TNP antibody response to TNP-HRBC, a T-dependent immunogen, was found to be lower in these mice as compared to LPS-responsive C3H/HeN mice, whereas the anti-TNP antibody response to TNP-Ficoll, a T-independent immunogen, was of the same magnitude in C3H/HeJ and C3H/HeN mice. An impaired helper activity of C3H/HeJ HRBC-primed spleen cells was demonstrated in a titration assay in which graded numbers of C3H/HeJ or C3H/HeN HRBC-primed spleen cells were added to cultures containing a constant number of unprimed spleen cells from either C3H/HeJ or C3H/HeN mice and the immunogen TNP-HRBC. The reduced helper T-cell activity of C3H/HeJ HRBC-primed spleen cells appears to be independent of macrophage defects, since C3H/HeJ and C3H/HeN macrophages were found equally effective in antigen presentation as evaluated by an in vitro antigen-specific T-cell proliferation assay. The difference in helper T-cell activity between these two substrains probably reflects a lower number and/or proliferation rate of antigen-responsive T cells in C3H/HeJ mice.     

Inhibition of B lymphocyte activation by interferon-gamma

Helper/inducer T cell clones specific for protein antigens and class II MHC determinants consist of two nonoverlapping subsets. One (called Th1) secretes IL 2 and IFN-gamma and the other (Th2) produces BSF1 upon stimulation with antigen or polyclonal activators. By using hapten-binding normal B cells and the B lymphoma line WEHI-279 as assays for B cell helper (maturation) factors, we have shown that Th2 clone supernatants (SN) induce differentiation to antibody secretion, whereas Th1 SN do not. The failure of Th1 SN to activate B cells is due to inhibitory effects of IFN-gamma, because it can be reversed by a neutralizing monoclonal antibody specific for IFN-gamma. Thus, in the presence of this antibody, even Th1 SN stimulate B cell maturation maximally. Conversely, recombinant IFN-gamma inhibits proliferation and differentiation of B cells induced by active Th2 SN. These results demonstrate that IFN-gamma is a potent inhibitor of B lymphocyte activation and can be distinguished from growth and maturation-inducing helper factors that are produced by both subsets of helper T cells.     

Differences in the regulation of humoral responses between mice infected with Trypanosoma cruzi and mice administered T. cruzi-induced suppressor substance

In the present study, the effects of Trypanosoma cruzi and the T. cruzi-induced serum suppressor substance (SSS) on antibody responses were compared. Although infection with T. cruzi led to an alteration in T cell helper activity and a reduced specific B cell precursor frequency, SSS did not have a similar effect on either of these cell populations. The characteristics of the altered T cell helper activity was further investigated, and it was found that helper activity appeared earlier in infected mice than in normal or SSS-suppressed mice, and less antigen was required for optimal elicitation of T helper cells in infected mice. The potency of T cell helper activity also was shown to differ, and in the order T. cruzi-infected 6E normal 6E SSS-suppressed mice. It was found that spleen cells from T. cruzi-infected mice elaborated more potent specific helper factors than spleen cells from normal or SSS-suppressed mice, but did not produce a detectable nonspecific helper factor in vitro. Finally, the addition of B cells from low-dose primed, T. cruzi-infected mice to cultures of normal spleen cells resulted in subnormal responses to the priming antigen (sheep erythrocytes) but not to another unrelated antigen (trinitrophenyl-haptenated Brucella abortus), whereas similarly sensitized B cells from normal or SSS-suppressed mice caused no such effect.     

In vivo restoration of T cell functions by human IL-1 beta or its 163-171 nonapeptide in immunodepressed mice

The immunorestorative capacities of human (hu) IL-1 beta or its synthetic fragment 163-171 (VQGEESNDK) were assessed in vivo in mice immunodepressed by aging, sublethal irradiation, or both. Subcutaneous administration of hu rIL-1 beta into immunodepressed animals immediately after carrier (horse red blood cells, HRBC) priming could restore to normal levels Th cell activity. This was measured as the ability of spleen cells from HRBC-primed mice to induce a hapten-specific antibody response in spleen cells from nonimmune mice in vitro stimulated with the hapten-carrier conjugate TNP-HRBC. In parallel, the ability of spleen cells from hu rIL-1 beta-treated immunodepressed animals to produce T cell growth factor activity upon in vitro mitogen stimulation was also increased significantly as compared to that of untreated mice and approached that of immunocompetent controls. The immunorestorative activity of hu rIL-1 beta on Th cell activity and T cell growth factor production could be mimicked by the synthetic nonapeptide 163-171 which, at the doses used, produced in most instances even greater effects than the whole protein. Although the optimal immunorestorative doses of the 163-171 peptide were several orders of magnitude higher than those of hu rIL-1 beta, the complete lack of IL-1-like inflammatory and toxic effects suggests that the synthetic hu IL-1 beta fragment may be successfully used as immunomodulating agent in the therapy of T cell immunodeficiencies.     

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.     

CD4+ subset regulation in viral infection. Preferential activation of Th2 cells during progression of retrovirus-induced immunodeficiency in mice.

Progressive lymphoproliferation and increasingly severe immunodeficiency are prominent features of a syndrome, designated mouse AIDS, which develops in susceptible strains of mice infected with the mixture of murine leukemia viruses, termed LP-BM5. Development of splenomegaly and lymphadenopathy, caused primarily by increases in B cell immunoblasts, requires the presence of CD4+ T cells and is assumed to be mediated by lymphokines produced by these cells inasmuch as progression of disease is markedly inhibited by treatment of infected mice with cyclosporin A. Studies of spleen cells from infected mice revealed spontaneous production of cytokines (IFN-gamma, IL-2, IL-4, IL-5, and IL-10) characteristic of Th0 (or a mixture of Th1 and Th2) T helper cells at 1 wk after infection. At later times, IFN-gamma and IL-2, characteristic products of Th1 helper clones, were expressed poorly, either spontaneously or after stimulation of cells with Con A. In contrast, IL-4, IL-5, IL-6, and IL-10, cytokines typically synthesized by Th2 cells, were produced in response to Con A or spontaneously through 18 wk post-infection. Increased serum IgE levels and enhanced IL-10 mRNA expression were consistent with expression of Th2 cytokines at biologically significant levels in vivo. Selective depletion of T cell subsets before stimulation with Con A showed that CD4+ T cells were the primary source of IL-2, IL-4, IL-10, and, to a lesser extent, IFN-gamma in spleens and lymph nodes of normal or infected mice. These results suggest that persistent activation of CD4+ T cells with the lymphokine profile of Th2 helper clones is responsible for chronic B cell stimulation, down-regulation of Th1 cytokines, and impaired CD8+ T cell function in mouse AIDS. This provides the first demonstration that, like many parasitic infections, viruses encoding potent antigenic stimuli can markedly affect the balance of Th subset expression.     

Enhancement of T helper2 response in the absence of interleukin (IL-)6; an inhibition of IL-4-mediated T helper2 cell differentiation by IL-6

Functional roles of interleukin (IL-)6 in T cell response were investigated. Mice deficient in IL-6 and wild mice were immunized with antigens (myelin oligodendrocyte glycoprotein or methylated BSA) and production of IL-4 and interferon (IFN)-gamma by regional lymph nodes was measured. IL-6 deficiency led to an enhancement of IL-4 and an inhibition of IFN-gamma production. Moreover, polyclonal stimulation of spleen T cells from unimmunized IL-6-deficient mice with anti-CD3 plus anti-CD28 antibodies (Abs) demonstrated an enhancement of T helper (Th)(2)responses. The presence of IL-6, however, augmented IL-4 production but it inhibited IFN-gamma expression by spleen T cells in response to polyclonal stimulation and by antigen-primed spleen T cells in response to re-challenge with the antigen. In contrast, the induction of spleen CD4-positive T cells into Th(2)cells in vitro by the anti-CD3 plus IL-4 was completely suppressed by exogenously added IL-6, whereas Th(1)differentiation of T cells by the anti-CD3 plus IL-12 was not inhibited by the presence of IL-6. Thus, these results indicate that IL-6 physiologically could modulate qualitative T cell response and suggest that it augments Th(1)responses partly through its inhibitory capability of IL-4-induced Th(2)differentiation of naive T cells.     

Long-term CD4+ memory T cells from the spleen lack MEL-14, the lymph node homing receptor.

We have characterized the surface phenotype and function of long-lived, Ag-specific memory CD4+ T cells generated in vivo by immunization with keyhole limpet hemocyanin (KLH). CD4+ T cells from the spleens of mice primed more than 2 mo previously with KLH, produced high levels of IL-2 and IL-3, and low levels of IL-4 and IFN-gamma in response to in vitro restimulation with specific Ag. The KLH-primed T cells mediated carrier-specific helper activity for the antibody production by NIP-primed B cells in secondary in vitro responses to NIP-KLH. Subsets of CD4+ T cells from KLH-primed mice were isolated on the basis of surface CD45RB (23G2) by magnetic separation and were examined for functional capacity in several assays of Ag-specific recall. Virtually all of the secretion of IL-2, IL-3, IL-4, and IFN-gamma in response to restimulation with Ag in vitro was associated with, and considerably enriched in, the CD45RB- subset of CD4+ T cells. Similarly, carrier-specific helper function and Ag-specific proliferation in vitro were also confined to the CD45RB-, CD4+ subset of T cells, confirming the previous association of this surface phenotype with memory Th cell activity. We also examined expression of the lymphocyte homing receptor, MEL-14 (gp90MEL), which is required for lymphocyte extravasation to peripheral lymph nodes and is present in high levels on naive T cells. MEL-14 positive and negative subsets of CD4+ T cells from long term KLH-primed mice were evaluated for Ag-specific memory function in terms of lymphokine production, Ag-induced proliferation, and helper activity. Each of these functions was associated exclusively with the MEL-14- subset of CD4+ T cells, which exhibited responses comparable to the CD45RB- subset. These data indicate that memory Th cell function in the spleen is contained within the MEL-14-, CD45RB- subset of CD4+ T cells and suggest that memory helper cells may have different patterns of recirculation from naive T cells.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. II. Mechanisms, specificity, and cellular analysis of 2,4,6-trinitrophenol (TNP)-specific cytolytic response priming by intravenous versus subcutaneous injection with TNP-modified syngeneic cells

We have examined the underlying mechanisms accounting for the enhanced in vitro TNP-specific cytotoxic T-lymphocyte (CTL) response following the parenteral injection of syngeneic hapten-modified lymphoid cells. Augmented CTL activity noted following parenteral injection (iv vs sc) of 2,4,6-trinitrophenol-modified syngeneic spleen cells (TNP-SC) is most apparent when limiting numbers of TNP-modified stimulator cells are used in the in vitro sensitization phase. Enhanced CTL responses seen following sc and iv priming is due to distinct mechanisms. Spleen and lymph node (LN) cells from sc primed mice were found to contain significant levels of radioresistant helper activity upon coculture with either viable normal spleen cells in bulk culture or with thymocytes as the source of precursor CTLs in a limiting dilution assay. The helper activity was found to be mediated by a Lyt 1+2- T cells. In addition, Lyt 2-depleted spleen and LN cells from sc primed BALB/c mice could restore the ability of tolerant spleen cells from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-injected BALB/c mice to generate TNP-specific CTLs. Conversely, Lyt 2-depleted spleen and LN cells from iv primed mice provided no measurable helper activity either in bulk culture or in the limiting dilution assay and did not restore the ability of TNBS-tolerant BALB/c spleen cells to generate TNP-specific CTLs. CTL priming via the iv route was found to be completely antigen specific as iv injection of either 2,4-dinitrophenol (DNP)- or fluorescein isothiocyanatel (FITC)-modified cells caused no enhanced CTL activity. Priming via the sc route exhibited a unique specificity pattern as it was shown that sc injection of both TNP-SC and DNP-SC, but not FITC-SC, resulted in enhanced TNP-specific CTL responses. CTL T-helper (Th)-cell induction via the sc route was correlated with (1) the presence of H-2 I region determinants on the inducer cells as the sc injection of TNP-modified erythrocytes led to no enhanced CTL responses or CTL Th activity (while iv injection of TNP-erythrocytes did lead to enhanced CTL responses without detectable helper activity) and (2) the detection of both hapten-specific T-cell proliferation and Interleukin 2 (IL-2) production upon restimulation in culture. We conclude that the sc injection of TNP-SC leads preferentially to an increase of specific Lyt 1+ helper activity, while iv injection leads preferentially to an apparent expansion of Lyt 2+ prelytic effector CTLs.     

Primary response of naive CD4(+) T cells to amino acid-substituted analogs of an antigenic peptide can show distinct activation patterns: Th1- and Th2-type cytokine secretion, and helper activity for antibody production without apparent cytokine secretion

Naive CD4(+) T cells differentiate into two types of helper T cells showing an interferon-gamma-predominant (Th1) or an interleukin-4-predominant (Th2) cytokine secretion profile after repeated antigenic stimulation. Their differentiation can be influenced by slight differences in the interaction between the T cell receptor (TCR) and its ligand at the time of primary activation. However, the primary response of freshly isolated naive CD4(+) T cells to altered TCR ligands is still unclear. Here, we investigated the primary response of splenic naive CD4(+) T cells derived from transgenic mice expressing TCR specific for residues 323-339 of ovalbumin (OVA323-339) bound to I-A(d) molecules. Naive CD4(+) T cells secreted either Th1- or Th2-type cytokines immediately after stimulation with OVA323-339 or its single amino acid-substituted analogs. Helper activity for antibody secretion by co-cultured resting B cells was also found in the primary response, accompanied by either low-level Th2-type cytokine secretion or no apparent cytokine secretion. Our results clearly indicate that dichotomy of the Th1/Th2 cytokine secretion profile can be elicited upon primary activation of naive CD4(+) T cells. We also demonstrate that the helper activity of naive CD4(+) T cells for antibody production does not correspond to the amounts of the relevant cytokines secreted.     

Proliferative and cytotoxic immune functions in aging mice. II. Decreased generation of specific suppressor cells in alloreactive cultures

The ability of spleen cells from aged C57BL/6 mice to generate specific suppressor cells in mixed lymphocyte cultures (MLC) against allogeneic H-2 antigens was investigated. The suppressor cells from young and old mice were assayed in parallel for their ability to inhibit the proliferative response and the generation of cytotoxicity in fresh MLC. Suppressor cell generation was found to be significantly decreased in 41% of aged mice (23 to 28 mo) as compared to young controls (3-8 mo). The suppressor cells were H-2-specific, radiation-resistant (1000 R), and Thy-1+; they did not function by lysing the fresh stimulators or responder cells, or by absorbing the interleukin 2 in the fresh cultures. Suppression required very small numbers of cells to be effective. It was concluded that the effect of aging was less marked on specific suppressor cell generation than on generation of cytotoxic T cells in the MLC. However, a third type of response studied, the proliferative response, was affected earliest and most severely.     

Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors

The role and induction requirements of helper T lymphocyte responses to herpes simplex virus type 1 (HSV-1) was examined. Splenocytes from mice that had been primed in vivo with infectious HSV-1 can be restimulated in vitro with live or partially UV-inactivated HSV-1 to generate high levels of herpes virus-specific cytotoxic T lymphocyte (CTL) activity. By comparison, naive splenocytes or splenocytes taken from mice primed with heat-inactivated HSV-1 failed to generate CTL after in vitro viral stimulation. In addition, infectious HSV-primed splenocytes can be rendered unresponsive to secondary in vitro restimulation by pretreatment with anti-Lyt-1 antiserum plus complement. Spleen cells were taken from mice that had been primed and restimulated in vivo with infectious HSV-1. Two days after the second priming, splenocytes were prepared and irradiated. These cells were capable of assisting in the generation of CTL to varying degrees in all of the above unresponsive populations of cells. The irradiated cells did not produce detectable levels of CTL activity when cultured alone with antigen. Also, if the irradiated splenocytes were treated with anti-Lyt-1 plus complement before their addition to cultures, all restorative activity was ablated. In contrast, irradiated splenocytes from mice that had been primed and restimulated in vivo with either heat-inactivated or UV-inactivated HSV-1 were unable to provide help to naive or helper-depleted cultures. The failure to supply helper activity appears not to involve the preferential activation of suppressor cells, as evidenced by cell mixing experiments and the addition of concentrated, antigen-stimulated spleen cell supernatant fluids to secondary anti-HSV-1 splenocyte cultures. Proliferative assays using interleukin 2- (IL 2) dependent cell lines as a measure of relative helper activity indicated that the inactivated forms of HSV-1 were incapable of effectively enlisting helper activity. These experiments therefore suggest that the observed failure of heat-inactivated or UV-inactivated HSV-1 preparations to induce anti-HSV CTL responses reflects the inability of the HSV-1-specific subset of helper T lymphocytes to recognize these forms of the antigen.     

The effect of irradiation on the antibody enhancing helper T cells

Helper T cells specific for sheep erythrocytes were generated in lethally irradiated mice. The helper effect exerted by these cells was determined in vitro in spleen cell cultures. Irradiation of the helper cells in vivo or in vitro with 1-9 Gy resulted in a bimodal relation between radiation dose and helper effect when measured either immediately or 6 hours after irradiation. The dose-effect curve became nearly linear when tested 20 hours after irradiation. These results indicated that the helper population consisted of about 80 per cent of cells inactivated shortly after irradiation, possibly due to interphase death, and a second type showing a more retarded cell death. The latter cells were able to exert a helper function during the interval between irradiation and cell death.     

Antigen presentation to Th1 but not Th2 cells by macrophages results in nitric oxide production and inhibition of T cell proliferation: interferon-gamma is essential but insufficient

The induction and role of nitric oxide (NO) during antigen presentation by macrophages to T helper (Th) cell subsets was examined. When cultured with Th1 clones, macrophage APC produced NO only in the presence of cognate Ag, which in turn suppressed T cell proliferation. IFN-gamma production by the activated Th1 cells was essential for the induction of NO. Th2 cells presented with the same cognate Ag did not induce NO production and proliferated uninhibited. Coactivation of Th1 and Th2 cells specific for the same Ag indicated that Th2 cells did not inhibit NO production, but were sensitive to NO induced by stimulated Th1 cells. Antigenic activation of Th2 cells in the presence of rIFN-gamma resulted in NO-mediated inhibition of proliferation. Th2 cells provided only a cell-associated cofactor, whereas Th1 cells secreted a soluble cofactor for IFN-gamma as well, i.e., TNF-alpha. Finally, a role for IFN-gamma and NO during immune responses was studied in spleen cells obtained from immunized IFN-gamma(-/-) mice. NO production and subsequent inhibition of Ag-specific proliferation ex vivo was observed only after the addition of rIFN-gamma. These studies suggest an IFN-gamma-dependent regulatory role for NO during Ag-specific Th cell activation involving macrophages, with obvious implications for Th subset-dependent immune responses in general.