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Background: Most reported human H5N1 viral infections have been severe and were detected after hospital admission. A case ascertainment bias may therefore exist, with mild cases or asymptomatic infections going undetected. We sought evidence of mild or asymptomatic H5N1 infection by examining H5N1-specific T-cell and antibody responses in a high-risk cohort in Vietnam., Methods: Peripheral blood mononuclear cells were tested using interferon-γ enzyme-linked immunospot T assays measuring the response to peptides of influenza H5, H3, and H1 hemagglutinin (HA), N1 and N2 neuraminidase, and the internal proteins of H3N2. Horse erythrocyte hemagglutination inhibition assay was performed to detect antibodies against H5N1., Results: Twenty-four of 747 individuals demonstrated H5-specific T-cell responses but little or no cross-reactivity with H3 or H1 HA peptides. H5N1 peptide-specific T-cell lines that did not cross-react with H1 or H3 influenza virus HA peptides were generated. Four individuals also had antibodies against H5N1., Conclusions: This is the first report of ex vivo H5 HA-specific T-cell responses in a healthy but H5N1-exposed population. Our results indicate that the presence of H5N1-specific T cells could be an additional diagnostic tool for asymptomatic H5N1 infection.

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

Dendritic cells (DC) carry antigen into lymph nodes where they may cluster with CD4 and CD8+ lymphocytes and activate both subsets in the initiation of immune responses. Since DC do not leave the lymph nodes in the efferent lymph they may die within the lymph nodes. Another possibility is that they are targets for cytotoxic T cells (CTL) when expressing appropriate epitopes. This possibility was tested in vitro using human peripheral blood DC to stimulate the development of primary CTL in response to HIV-1 or one of its T-cell epitopes (e.g. env 111-126) and secondary CTL in response to type A influenza virus. Pooled CTL generated during six day cultures in 60 replicate 20 microliters hanging drops were tested in a conventional CTL assay. The HIV or HIV peptide stimulated CTL lysed HIV infected DC while the influenza-virus induced CTL killed DC targets infected with this virus. DC were not lysed significantly until they had been exposed to virus for 2-3 days and thus are not highly susceptible to lysis. However, killing of DC after 2-3 days infection with virus may be a feedback mechanism for removing antigen presenting cells after they have stimulated T cell responses. Removal of persistently infected DC by CD8+ CTL may also contribute to the reduction in DC numbers observed in blood and skin in HIV infection.

We have examined human CD4+ T-cell recognition of influenza A/Beijing/32/92 (H3N2) virus hemagglutinin following influenza virus HANA subunit vaccination. CD4+ T-cell repertoires were dominated by recognition of epitopes located in conserved regions of the molecule, in a major histocompatibility complex class II haplotype-dependent manner, analogous to that observed following natural infection.

We have previously demonstrated that intranasal (i.n.) administration of an immunodominant peptide (p1-111-139) derived from the house dust mite (HDM) allergen Der p 1 inhibits antigen-specific CD4+ T cell responses in H-2b mice. Here we report that i.n. peptide induced a rapid but transient activation of MHC class II restricted CD4+ T cells that peaked 4 days after peptide treatment and was of similar magnitude to that induced by parenteral immunization with antigen in adjuvant. During the early phase of the response lymph node and splenic T cells secreted a range of lymphokines when re-stimulated in vitro with p1 111-139; however, by day 14 IL-2 and IFN-gamma secretion by T cells were down-regulated. Mice deficient in CD8+ T cells became tolerant by i.n. treatment with peptide, suggesting that CD8+ T cells are not involved in down-regulating the CD4+ T cell response. Rechallenging mice with a single dose of p1 111-139 21 days after the initial treatment elicited a further transient T cell response, which was subsequently down-regulated over time. Although the i.n. peptide induced a strong transient CD4+ T cell response, only low levels of peptide-specific antibodies were detected either after the initial or subsequent i.n. exposures to p1 111-139. Our findings address the mechanisms underlying peripheral T cell tolerance following i.n. administration of a high dose of immunogenic peptide and have implications for understanding the consequences of peptide immunothearapy.

The human CD4+ T-cell repertoire of responses to hemagglutinin (HA) of influenza virus A/Beijing/32/92 was examined 3 to 6 months after natural infection by using a panel of 16-mer peptides overlapping by 11 residues. Short-term CD4+ T-cell lines were derived by using full-length HAs of virus A/Beijing/32/92 from 12 unrelated, major histocompatibility complex (MHC) class I and II haplotyped adults with a history of influenza in November and December 1993 and from 6 adults with no history of influenza during the preceding 4 years but who responded to HA. In contrast to recent murine studies, the human CD4+ T-cell repertoire of responses was dominated by the recognition of highly conserved epitopes. The HA2 subunit, widely regarded as nonimmunogenic, induced strong responses in every donor. This resulted in functional cross-reactivity among influenza A viruses. Our study included one pair of unrelated donors expressing identical HLA DRB1 and DQB1 alleles and two pairs of donors sharing low-resolution MHC class II types. These pairs responded to identical peptides; furthermore, clearly identifiable patterns of response were seen in donors sharing single class II haplotypes, irrespective of the presence of other alleles and exposure history. Two conserved regions which induced responses in 17 of 18 donors were identified (residues 295 to 328 and 407 to 442). Possible implications for cross-reactive T-cell vaccines are discussed.

Virus-specific cytotoxic T-cell (Tc) clones can lyse target cells in vitro in the presence of their specific peptide epitopes. The lytic potency of murine influenza nucleoprotein (NP)-specific Tc clones was investigated after observing that target cell killing was reduced in the presence of high (greater than 0.2 microM) concentrations of specific NP peptide antigen. Following incubation of Tc for 16 hr in the presence of a range of peptide concentrations, two effects were observed; (i) a peptide dose-dependent mortality of Tc, which has been attributed to self-lysis by clonal Tc in the presence of specific peptide; (ii) and a reduced ability to specifically lyse NP-expressing target cells whilst retaining lectin-dependent lytic activity in the surviving Tc. This functional down-regulation was reversible after 24 hr incubation in the absence of peptide. Toxic effects were excluded, since inhibition of specific target lysis by Tc was mediated only be pretreatment with specifically recognized peptide.

The mechanism by which cytotoxic T-lymphocytes (Tc) induce the death of specific target cells is still controversial. We have used quantitative cytochemical methods to distinguish the metabolic activities of the target cells from those of the Tc, even when they are attached to each other. Early events following Tc-P8(15) target cell interaction were first, increased glucose 6-phosphate dehydrogenase activity and second, labilization of the lysosomes within the target cell: these changes could be mimicked, in part, by polyamines and could be inhibited by inhibiting ornithine decarboxylase (ODC) activity. The crucial role of ODC in the chain of events that led to cytolysis in this particular experimental system was shown first, by measuring ODC activity directly and secondly, by the inhibition of cytolysis by the presence of a selective inhibitor of ODC activity.

Recombinant vaccinia viruses containing the 22-kilodalton protein (matrixlike or 22K protein) or phosphoprotein gene from respiratory syncytial virus were constructed. These recombinant viruses expressed proteins which were immunoprecipitated by appropriate respiratory syncytial virus antibodies and comigrated with authentic proteins produced by respiratory syncytial virus infection. The new recombinant viruses (and others previously described containing the attachment glycoprotein, fusion, or nucleoprotein genes of respiratory syncytial virus) were used to infect target cells for cultured polyclonal cytotoxic T lymphocytes generated from the spleens of BALB/c or DBA/2 mice primed by intranasal infection with respiratory syncytial virus. Respiratory syncytial virus-specific cytotoxic T lymphocytes (CTL) showed strong Kd (but not Dd)-restricted recognition of the 22K protein. As previously reported, the fusion protein and nucleoprotein were both seen by CTL, but recognition of these proteins was comparatively weak. There was no detectable recognition of other respiratory syncytial virus proteins tested (including phosphoprotein). 22K protein-specific splenic memory CTL persisted for at least 11 months after infection of BALB/c mice. Priming BALB/c mice with recombinant vaccinia virus containing the 22K protein gene induced respiratory syncytial virus-specific memory CTL at lower levels than that previously reported following infection with a similar recombinant containing the fusion protein gene. These data identify the 22K protein as a major target antigen for respiratory syncytial virus-specific CTL from H-2d mice primed by respiratory syncytial virus infection.

Several CD4+ Th1 clones specific for influenza haemagglutinin or nucleoprotein were transferred into syngeneic mice after intranasal influenza infection to examine whether they accelerate viral clearance in vivo similarly to CD8+ cytotoxic T cells. We observed changes in functional properties of the CD4+ clones in vitro and variable effects on the course of infection in vivo. While some clones resulted in more rapid virus clearance, others had no protective effect, but rather exacerbated illness symptoms. Our results reflect problems in the in vivo use of CD4+ T cell clones maintained in long-term culture. Their IL-2 and IL-5 release and cytolytic activity varied, while IL-3 and gamma-IFN production as well as DTH induction were more stable. CD4+ T cells primed by infection became cytolytic only after prolonged culture. The data point to the fine balance between exacerbation of disease and protection by CD4+ T cells.

The recognition of "self determinants" by an H-2Kk-restricted cytotoxic T (Tc) cell clone (K5) has been investigated as follows: (a) differential inhibition of cytotoxicity by several monoclonal antibodies directed to determinants on the H-2Kk molecule of the target cells and (b) recognition and lysis of target cell variants of cell line LDHB, which have lost the majority or all of the serological determinants defined by the inhibiting antibodies while still expressing an H-2Kk molecule. Such variant cells infected with influenza virus were effectively recognized by Tc cell clone K5, whereas a target cell line, which lacks the Kk molecule, was not lysed. The results suggest; (a) that virus-specific Tc cell see "self" in a manner distinct from the recognition of serological determinants by B cell and (b) that antibody inhibition indicates conformational closeness, but not identity, of the class I determinant seen by T cells.

An ammonium deoxycholate fraction from bromelain-treated influenza A virus was highly enriched for virus nucleoprotein and contained residual haemagglutinin (NP/HA). The preparation did not contain detectable levels of matrix or neuraminidase proteins and was free of infectious virus. NP/HA effectively primed mice for cytotoxic T cells which lysed syngeneic cells infected with any type A influenza virus. Furthermore, NP/HA generated A-type virus cross-reactive cytotoxic T cells when added in vitro to spleen cells from mice previously primed with infectious influenza A virus. These properties imply that NP/HA has potential as a vaccine for heterotypic influenza A immunity.

This report describes further attempts to define the nature of the parasite product(s) responsible for the extensive changes in lymphoid tissue in mice during infection with Trypanosoma brucei. As previously described, potent mitogenic and immunosuppressive effects are induced by a trypanosome-derived crude membrane fraction in vivo. There was no enrichment in these activities when purified parasite surface membranes were used. Mitogenic activity can be recovered from soluble trypanosome material only when it is incubated with peritoneal macrophages before transfer into syngeneic recipients. Thus, by encouraging association with a critical target cell, soluble parasite products can be studied, and their active components can be separated by conventional methods. Preliminary fractionation of high-spin trypanosome supernatant over Sepharose 4B confined the mitogenic activity to the high-molecular-weight fraction, which is a macromolecular complex of proteins, glycoproteins, and lipid. Extracted lipid from this material was able to significantly suppress a primary immunoglobulin G anti-sheep erythrocyte response. The activity was periodate sensitive and pronase resistant. The use of macrophages in vitro may be a general method whereby important biological activities lost as a result of fractionation procedures can be recovered and the active components studied in greater detail.

A considerable amount is known about Ig biosynthesis by mature plasma cells, which form large amounts of Ig for secretion from the cell. A brief summary is given of the formation of light (L) and heavy (H) chains by polyribosomes aligned on the endoplasmic reticulum and the rapid assembly of the chains into 7S molecules (H2L2) by disulphide bonding. There is a time-ordered secretion from the cell of 7S Ig molecules; the polymeric forms of Ig, ie, IgM and IgA, are formed from monomers by disulphide bond interchange and J chain incorporation at the time of secretion. Myeloma cells from mouse and man have proved very useful in this type of study but such malignant cells show many defects in regulatory mechanisms; therefore, no conclusions can be drawn about normal control mechanisms without analysis of lymphoid tissues from normal or immunized animals. The pattern of Ig synthesis by the mature cell contrasts with that by small B lymphocytes which form 1/50 to 1/100 the amount of Ig produced by mature cells. Most of the small lymphocyte Ig is associated with the cell surface, and in IgM-producing cells the surface receptors are 7S monomer subunits of IgM. Such receptors turn over slowly (24-48 hours); they may be gradually shed from the cell surface but the small lymphocyte does not actively secrete Ig. Antigen- and cell-cell interactions stimulate small B lymphocytes to divide and mature into Ig-secreting cells. Little is known about the associated intracellular events, but preliminary data on lipopolysaccaride-stimulated mouse spleen cells indicate that transcription of m-RNA for H-chain mirrors the kinetics of DNA synthesis. A translational block then occurs during cell maturation and there is a lag of at least 24 hours before Ig production rises sharply and reaches peak levels.

Virus specificity of T-helper cells for the humoral antibody response to influenza A viruses was studied with a hapten-carrier secondary adoptive transfer system, using whole virus, or viral components inserted into liposomes as carrier with B cells primed to DNP human gamma globulin. Evidence was obtained for two distinct T-helper cell populations from mice primed by influenza infection: a T-helper cell cross-reactive for all type A influenza viruses and a second T-helper population specific for the variant haemagglutinin. In vivo the virus cross-reactive T helpers recognized whole virus, but did not recognize isolated surface glycoproteins or internal virus proteins.

This study reports the examination of in vivo and in vitro properties of an antigen-dependent murine cytotoxic T cell (Tc) clone T5/5 specific for type A influenza virus. This clone differs morphologically and in its migratory pattern and biological properties from a previously examined anti-influenza Tc clone L4 which grew in T cell growth factor independently of antigen. Unlike Tc clone L4, T5/5 cells do not release significant amounts of immune interferon on contact with influenza-infected target cells nor do they limit virus replication in vivo, although they efficiently lyse influenza-infected target cells and can release interferon in the presence of concanavalin A. Thus individual Tc clones vary in function and further work is required to establish the properties of Tc that are associated with host protection against virus infection.

Little is known of the role of T-cell mediated immune responses in the clearance and pathogenesis of respiratory syncytial virus (RSV) infection. In this study, we established persistent pulmonary RSV infections in athymic nu/nu BALB/c mice or immunodeficient irradiated BALB/c mice, and examined the patterns of virus clearance following adoptive transfer of splenic memory T cells. Primed T cells transferred between Day 5 and Day 8 of infection will clear lung RSV from both nu/nu mice and irradiated mice within 10 days of transfer. Partially purified Lyt 2+ T cells are more effective than L3T4+-selected T cells. No RSV-specific serum antibody could be detected, suggesting that clearance is by an antibody-independent mechanism. In contrast, delayed (Day 14) transfer of primed L3T4+-selected cells clears lung RSV from nu/nu mice, and this correlates with RSV-specific serum antibody production. Clearance is not seen following Day 14 transfer of total primed T cells or T cells selected for the Lyt 2+ subset.

C57BL/6 mice generate influenza-specific cytotoxic T cells (Tc) which are predominantly restricted to Db. The induction of a Kb-restricted response in H-2b mice has been controversial. We show here that the magnitude of Kb-restricted anti-influenza Tc responses is affected by the type of stimulator cell. Both in vivo and in vitro, secondary responses in the spleen show strong selection for Db-restricted Tc. However, limiting dilution analyses show that intranasal (i.n.) influenza infection primes Kb- as well as Db-restricted Tc in C57BL mice at a ratio of 1:2--but different cell types acting as stimulator cells vary in their ability to induce secondary Kb-restricted influenza-specific Tc (B lymphoblasts greater than macrophages greater than spleen cells greater than T lymphoblasts). Thus, the site of infection and antigen presentation can determine the MHC restriction of T-cell responses.

African trypanosomiasis is accompanied by profound general immunosuppression. The experiments described here were designed to characterize the contribution of macrophages to the immune pathology of this disease. We used peptone-stimulated, uninfected mice and injected them intraperitoneally with lethally irradiated and 35S-labeled Trypanosoma brucei and parasite-specific antisera. Peritoneal macrophages were thus induced to take up in vivo a defined number of trypanosomes. After the phagocytosis of parasites, macrophages were transferred into uninfected syngeneic mice, where they mimicked some of the important immunological changes normally associated with active trypanosome infection: (i) splenic background plaque-forming cells increased nonspecifically and (ii) the specific immune response to sheep erythrocytes was either enhanced or suppressed, depending on the timing of the antigen challenge: priming simultaneously with the transfer of trypanosome-containing macrophages enhanced immune responsiveness; in contrast, if parasite-containing macrophages were transferred and recipient mice were primed 4 days later, the immune response was suppressed. A contribution of suppressor T cells was ruled out by the treatment of peritoneal exudate cells with anti-Thy 1.2 and complement before transfer into recipient mice. The results indicate that macrophages are key cells in the mediation of parasite-induced immune dysfunction.

Infective influenza virus primes mice and increases at least ten-fold the level of splenic cytotoxic T-memory and precursor cells in comparison with normal mice. Intranasal virus infection or intraperitoneal injection of infective virus results in frequencies of 1-2 x 10(-4) cytotoxic T-cell precursors in spleen as determined by limiting dilution assays. With both types of immunization, T-helper cells amplifying the generation of T-killer cells are limiting, and optimal clone frequencies depend on addition of excess T-helper cells. We find that at least part of the T-helper cells amplifying the generation of cytotoxic T cells are cross reactive for the type A influenza viruses and therefore have a similar virus specificity to type A influenza-specific cytotoxic T cells (tc). Help for T-killer cells can be replaced by supernatants derived from Concanavalin A-stimulated rat spleen cells, but presence of antigen is still required to stimulate the Tc precursor or memory cells before they respond to antigen non-specific T cell-growth factor(s) present in the stimulated rat spleen cell medium.

Direct visualization of the events resulting from LPS stimulation of mouse spleen cells in vitro was achieved by characterizing the cells during four days of culture for morphology, Ig and theta surface markers and autoradiography after [3H] thymidine uptake. The changes observed were related to biochemical parameters such as incorporation of [3H] thymidine into DNA, Ig biosynthesis and secretion. Two pathways of B lymphocyte differentiation were observed: a) the generation of a large number of small B lymphocytes with high density of surface Ig but no internal pool detectable by immunofluorescence, and b) the maturation of a very small proportion of cells with a large intracellular pool and the ability to secrete Ig. Both cell types arise from dividing blast cells, either physically separated or traced by pulse chase experiments with [3H] thymidine. We discuss whether this duality is caused by the triggering of different B cell subpopulations at different developmental stages, preprogramed to one or the other pathway or whether the final direction of development depends on the microenvironment of individual dividing cells.

Monoclonal antibodies (H100-30/3 and 11-4.1) to H-2k were used to study H-2 antigen expression and characteristics of the H-2 antigen-antibody interaction at the cell surface. Studies with radiolabelled F(ab')2 and Fab' fragments of 11-4.1 antibody confirmed that monoclonal IgG binding to cells is directly proportional to the number of H-2 sites and shows a high proportion of monovalent binding over a wide range of concentrations. Scatchard plots showed no difference in the binding affinity constant (Ka) of a given monoclonal antibody on lymphoblasts from various H-2k F1 and congenic strains, but only in the number of antigenic sites per cell. F1 (k x d) lymphoblasts show 1 x 10(5) H-2k sites/cell, about 50% of the expression in homozygotes. Dk expression in C3H.OH is 1.4 x 10(4) sites/cell. While normal cells appear to have a constant amount of H-2 (2-3 x 10(5) sites/cell), BW thymoma cells show unstable H-2 expression, having an average of five times fewer H-2 sites per cell when grown in vitro as compared to in vivo growth. Another BW cell surface marker, Thy-1.1, does not fluctuate in parallel with H-2. The 30/3 and 11-4.1 antibodies bind to topologically distinct sites on H-2Kk. The binding of these antibodies can be perturbed differentially: paraformaldehyde fixation of cells abolishes binding of 11-4.1 antibody but not of 30/3 antibody; increasing temperature increases the Ka of 30/3 antibody binding but decreases the Ka of 11-4.1 antibody binding to cells.

(CBA x C57BL/6)F1 antisera prepared by injecting F1 mice with CBA T lymphocytes or CBA anti-C57BL/6 alloantibodies were tested for their ability to inhibit the mixed leukocyte culture (MLC) response. Presence of antiserum throughout the culture period in the absence of complement did not have any effect on the MLC response. Treatment of CBA responder cells with F1 antiserum and complement prior to the culture specifically inhibited the MLC response. Specificity of the suppression was ascertained; absorption of the F1 sera with F1 or C57BL/6 spleen cells did not remove the suppressive factor, whereas absorption with CBA spleen cells did so. F1 antiserum treatment left intact the response to third party alloantigens (DBA/2). Immunabsorbent columns with alloantibody of corresponding specificity removed the suppressive factor from anti-T cell sera as well as from antisera to alloantibody. The data suggest that the circulating alloantibody population contains molecules which share idiotypic determinants with surface receptors on T cells recognizing the same alloantigens.

Previous reports have described profound effects on the function of the lymphoid system, especially the spleen, in mice infected with Trypanosoma brucei. This study provides further evidence of major change in the cell populations of the blood, peritoneum, and bone marrow, but shows that at least some of the stem cells of the bone marrow survive the damage caused by trypanosomes and retain their ability to repopulate the animal. In these infected mice the initial parasitemia was terminated by day 11 and was followed by a subpatent period of approximately 7 days before a final, lethal parasitemia occurred. Lymphopenia preceded the initial and final waves of parasites in the blood, and there was a marked increase in circulating neutrophils and large mononuclear cells for 1 week after the termination of the first wave of bloodstream parasitemia and during the final lethal parasitemia. Dividing macrophages were detected in the peritoneum only briefly during week 1 of infection, but the total number of peritoneal cells was increased from day 8 until the mice died. The bone marrow is severely stressed by the parasite infection. Total cell numbers and spleen colony-forming cells in the bone marrow were profoundly depleted during the resolution of the first parasitemia, but both these parameters largely recovered during the subpatent period before the mice were killed by the disease. Immune function was restored gradually after treatment with Berenil late in infection. We conclude that the progenitors of lymphocytes as well as the mature cells are affected by trypanosomes, but that some of the early bone marrow stem cells escape and rapidly repopulate the peripheral organs upon removal of the parasites.

A series of 16 H-2b-restricted, A influenza virus-specific cytotoxic T-cell clones are described and characterized. One is Kb restricted, the others Db restricted. The factors governing Kb or Db restriction patterns seen in the mixed populations from which clones are derived are investigated. The Kb-restricted clone does not recognize Kb mutant bml and influenza and all 15 Db-restricted clones do not recognize Db mutant bml4 and A influenza virus; these results are discussed in the light of findings in a variety of other viral systems. Representative Kb- and Db-restricted clones were used to assess the functional properties of cloned cosmids containing either Kb or Db genes expressed in transformed L-cells (k haplotype). The expression products of both cosmids functioned efficiently as mutually exclusive restriction elements for A influenza virus recognition.

Subunit and intact influenza A virus vaccines have been compared with infectious virus in a mouse model for their ability to induce memory for cross-reactive cytotoxic T cell responses and to protect mice from challenge with different subtypes of influenza A virus. There is an overall correlation between secondary cytotoxic T cell responses and cross-protection. The most long-lasting and successful cross-protection was observed after intranasal infection with influenza virus A/X31 (H3 N2) that replicates efficiently in mice and induces high levels of memory for cross-reactive cytotoxic T cell responses. Short-lasting cross-protection and low levels of T cell-mediated cytotoxicity were associated with infection by A/USSR (H1 N1) virus, that replicates to lower titers in mice, or after multiple injections of inactivated whole virus vaccine. No cross-protection to challenge with heterologous influenza virus was detectable after 1-2 injections of HANA influenza subunit vaccine which failed to prime hosts for cytotoxic T cell responses. These findings may have important implications for vaccination strategy. If cytotoxic T cells play a role in the protection of humans from influenza, live attenuated vaccines should be considered instead of the currently recommended inactivated virus or subunit vaccines.

We have studied the release of immune interferon (IFN-gamma) by influenza-specific cytotoxic T-cell (Tc) clones. IFN-gamma release is entirely dependent on specific antigen recognition or mitogen treatment and correlates inversely with the growth rate of the clone, while no differences in cytotoxic activity can be discerned at the different stages of Tc maturation. Although the mitogen Con A provides a more powerful stimulus for IFN release by Tc clones, specific antigen leads to a more rapid secretion, starting within 2 hr of contact with Tc clones and their specific targets. This may be of significance in an infection, providing a quick, but localized, mechanism to prevent viral spread. We also examined whether ligand interactions with T-cell surface glycoproteins Lyt-2 or LFA-1, important in Tc recognition, affected IFN release. Monoclonal antibodies to both Lyt-2 and LFA-1 block specific target cell lysis of Tc clone BA4, but do not affect Tc clone T9/5. This latter finding adds LFA-1 to the list of T-cell surface components which are not always essential for target cell recognition. Antibody to Lyt-2 blocked antigen-induced IFN-gamma release by all Tc clones studied, whilst two monoclonal antibodies to LFA-1 had little or no effect. Thus, the Lyt-2 molecule plays a role in the regulation of IFN secretion.

African trypanosomiasis in mice leads to profound changes in lymphoid tissues. In an attempt to define the nature of the trypanosome stimulus, we have studied the effect of radio-attenuated trypanosomes and their subcellular fractions in vivo. We find that relatively low doses of irradiated Trypanosoma brucei S42 injected into (CBA/H x C57B1/6)F1 mice mimicked the previously reported effects of infective parasites. 2 x 10(7) irradiated trypanosomes caused a greater than two-fold increase in spleen weight accompanied by a roughly 10-fold increase in background plaque forming cells (PFC) to sheep red blood cells (SRBC). The primary response to SRBC was significantly enhanced when priming was carried out on the day of trypanosome injection, but significantly suppressed when carried out 3 days later. Disruption of trypanosomes by freeze-thawing did not destroy their mitogenic or immunosuppressive activities. A membrane fraction collected by high speed centrifugation (150 000 x g) after removal of larger organelles at 12 000 x g retained both mitogenic and suppressive activities. The high speed supernatant lost the ability to enhance background PFC, but still caused partial immunosuppression with a much lower potency than the membrane pellet. Whether immunosuppression and enhanced PFC levels relate to the same parasite product is not clear as yet, but both effects can be ascribed to a membrane fraction of the parasite.

T cells and their subpopulations have many different functions important for (i) the regulation of immune responses through the release of antigen non-specific lymphokines and (ii) as effector cells to rid the host of intracellular pathogens, be they bacteria, parasites or viruses. In this short summary only a few features of T-cell function can be summarized, and studies in virus infection will serve as illustrations.

Local administration of nucleoprotein purified from X31 (H3N2) influenza A virus primed for A virus cross-reactive cytotoxic T cells and resulted in substantial protection (75%) of mice from a lethal challenge with the heterologous mouse-adapted A/PR/8/34 (H1N1) virus. By following the course of a lethal virus challenge we found that nucleoprotein priming did not prevent virus infection but rather aided recovery. Nucleoprotein-primed mice suffered initial symptoms of infection, i.e. weight loss and surface temperature changes, but started to recover after approximately 7 days. We suggest that such heterotypic protection can be attributed to priming of A virus cross-reactive cytotoxic T cells.

We have examined the function of class I MHC-restricted cytotoxic T cells in experimental respiratory syncytial virus (RSV) infection of BALB/c mice by transfer of T cell line MJC-A2 and CTL clone E8a into RSV-infected mice. The T cell line cleared pulmonary RSV infection within 5 d in persistently infected gamma-irradiated mice, but caused acute respiratory disease. This was only seen in infected mice and was often lethal after transfer of greater than 3 x 10(6) CTL. Lower numbers of CTL produced less severe disease but still cleared lung RSV, albeit over a longer time course (up to 10 d). Clearance of lung RSV in immunocompetent mice by the T cell line and CTL clone was again accompanied by acute and sometimes lethal respiratory disease. Bronchoalveolar lavage showed severe lung hemorrhage and frequent neutrophil efflux in mice with CTL-augmented disease.

The nature of Ig receptors carried by lipopolysaccharide (LPS)-stimulated mouse spleen B cells was analyzed by surface iodination, direct antiserum precipitation and polyacrylamide gel electrophoresis. LPS activation led to a rapid decrease of surface IgD to 20 % and 80 % of the original level on days three and five of culture, respectively. Efficiency of iodination of cells doubled after culture but the proportional radioactivity in IgM reace of cells, but only in small amounts (1 % of total surface Ig) and this expression of IgG remained constant during 5 days of culture. We could definitively identify gamma-chains on the surface of cells, but only in small amounts (1% of total surface Ig) and this expression of IgG remained constant during 5 days of culture.