Local and peripheral cell-mediated immune response to influenza virus in mice

Presentation of different influenza virus antigens generates different immune responses. Intranasal immunization with either live (VA) or formalin-inactivated (VF) A/PR/8/34 (HON1) influenza virus induced local as well as peripheral cell-mediated immune response (CMI), as evidenced by elevation in 3H-thymidine incorporation. Cell-mediated immune response was detected as soon as 24-48 hr following the application of VA and 4-5 days following VF. Cell-mediated immune response in both instances peaked on the 12th day and disappeared between 16 and 20 days after application. Local CMI response was threefold higher after immunization with VA (SI = 28.6) than with VF (SI = 9.4), while VF induced higher peripheral response (32.0 vs 17.7). The mononuclear cell population in the lungs increased, correlating with a rise in the stimulation index (SI). The percentage of IgA surface-bearing B lymphocytes was significantly higher following IN administration of VA, but not following VF instillation. This corroborated the finding that VF failed to induce local antibody response in the lungs in spite of its capacity to stimulate humoral antibody and CMI responses. Mice immunized intramuscularly with both viral preparations developed a fair humoral antibody response without detectable CMI (peripheral or local).     

Cell-mediated immune responses to the hemagglutinin and neuraminidase antigens of influenza A virus after immunization in humans.

Humoral and cell-mediated immunity (CMI) were evaluated in groups of school children after immunization with inactivated influenza virus vaccines. A conventional biphasic strain (H3ChN2Ch) of Port Chalmers influenza virus (X-41), a recombinant influenza virus specific for the neuraminidase antigen (Heq1N2Ch) of Port Chambers influenza A virus (X-42), and a placebo were employed for immunization. The techniques of hemagglutination inhibition and neuraminidase inhibition were used to determine serum antibody titers. The CMI responses were evaluated by the in vitro lymphocyte transformation assay employing HavN2Ch, Heq1Neq1, H3ChNeq1, and H3ChN2Ch influenza A virus strains as stimulants. Specific HAI antibody and CMI responses to H3Ch were observed in X-41 but not in X-42 vaccinees. Specific anti-neuraminidase antibodies and CMI responses to N2Ch were manifested by both X-41 and X-42 vaccinees. Immunization with the placebo resulted in no influenza-specific immune responses. The CMI response was first detectable 10 days after immunization and then declined. These observations demonstrate the induction of CMI responses to the HA and NA influenza surface antigens after immunization. These responses may be important in antiviral immunity and the recovery from influenza infection.     

Hemagglutinin and neuraminidase specific cell-mediated immune responses to influenza A virus following immunization in guinea pigs

Groups of guinea pigs were immunized with different inactivated recombinant influenza A viruses including H3ChN2Ch, Heq1N2Ch, H3ChNeq1 or uninfected allantoic fluid. Employing hemagglutination and neuraminidase inhibition tests, an in-vitro lymphocyte transformation (LTF) assay, and rosetting techniques for the separation of lymphocytes, influenza hemagglutinin (HA) and neuraminidase (NA) specific antibody and cell-mediated immune (CMI) responses were evaluated. Inactivated H3ChN2Ch, H3ChNeq1, HavN2Ch, and Heq1Neq1 recombinant influenza viruses were used as test antigens. Following immunization the CMI and antibody responses to influenza were characterized by the induction of specific LTF and antibody activity to homotypic HA or NA antigens but not to heterotypic HA or NA antigens. The temporal kinetics of the antibody response to influenza antigens was characterized by a prompt onset being initially detected at 1-2 weeks and reaching peak titers 3-4 weeks after immunization. Influenza specific LTF responses were first detected one week after immunization and declined to minimal responses at eight weeks. T-lymphocytes but not B-lymphocytes were capable of in-vitro recognition of the HA and NA antigens. After recognition the subsequent in-vitro lymphoproliferation was shown to involve both T and B lymphocytes.     

Relationship between cutaneous delayed hypersensitivity and cell-mediated immunity in vitro responses assessed by diphtheria and tetanus toxoids.

Cutaneous delayed hypersensitivity (CDH) testing with microbial antigens in man is thought to reflect the status of cell-mediated immunity (CMI). We have evaluated diphtheria-tetanus (DT) and tetanus (T) toxoids by comparing the CDH response with in vitro parameters of CMI: lymphocyte deoxyribonucleic acid (DNA) synthesis, leukocyte inhibition factor (LIF) in 5 immunized adults, and lymphotoxin release in 2 adults. Cord blood lymphocytes were used as controls for each assay. A dose response with both toxoids was used to compare the CDH reaction with each in vitro assay, establishing the maximum response and threshold dose which gave a positive response. All subjects had a positive CDH response to both antigens (≥5 mm induration at 48 hr), positive DNA synthesis (stimulation index ≥3), LIF release (migration ≤80%), and lymphotoxin production, while cord blood lymphocytes were usually negative to all in vitro assays. No consistent quantitative relationships between CDH reactions and in vitro CMI responses were seen. Threshold antigen dose data revealed that DNA synthesis was approximately ten times as sensitive an assay as CDH, and 10⁵ times as sensitive as the LIF technique. No difference in sensitivity was noted between DT and T toxoids. Three subjects re-evaluated 16 mo after the initial study showed positive CDH and CMI responses to tetanus toxoid, although the antigen dosage required varied considerably. We conclude that the CDH response with either toxoid in the concentrations used is a good indicator of CMI in the immunized individual. Although recommended starting antigen dose is given for each assay, a dose response for each assay must be performed to adequately evaluate the CMI responsiveness to a test antigen.     

Cytomegalovirus-directed lymphocyte reactivity in healthy adults tested by a CMV-induced lymphocyte transformation test.

A cytomegalovirus (CMV) induced lymphocyte tranformation test (LTT) performed as a microtechnique assay is described. Investigations were done in a group of twenty-five healthy adults with normal lymphocyte reactivity to phytohaemagglutinin and previously encountered antigens. Lymphocyte reactivity was established in CMV-seropositive persons using heat-inactivated cell-free CMV strain AD 169 in an optimal stimulating dose of 10(1-5) TCID 50. Positive CMV-LTT results were shown to be induced by purified inactivated CMV. Most persons (twelve out of seventeen) with antibodies against CMV-induced late or virus-structure antigens (LA) showed positive (stimulation index greater than 2-0) CMV-LTT results. Magnitude of CMV-LTT responses was positively related to the CMV-LA Ab titres (P less than 0-01). All persons (six) with antibodies against CMV early or non-structural antigens showed positive CMV-LTT results and highest stimulation rates were found in this group. Generally CMV-seronegative persons were non-responsive. Only in one case (LA Ab titre 10) a borderline positive response (stimulation index 2-3) was seen. The CMV-LTT was shown to have virus-specific properties because it was not related to simultaneously performed LTTs with Epstein-Barr virus and Herpes simplex virus in the same persons. It is concluded that CMV-LTTs are useful virus-specific in vitro techniques for the study of CMV-directed lymphocyte reactivity. Its possible relationship to other cell-mediated immunity tests and its practical applications are discussed.     

The antigens and nucleotide sequences of influenza A and B viruses in the lymphocytes of human peripheral blood]

Markers of influenza A and B viruses (antigens of hemagglutinin and specific nucleotide sequences) were detected in lymphocyte preparations from normal subjects. The rate of detection of the antigens and specificity (type and subtype appurtenance) of the markers correlated with the influenza epidemic situation. Lower titers of antibodies to the virus whose antigens were present in lymphocytes were observed.     

Respiratory Tract Cell-Mediated Immunity: Comparison of Primary and Secondary Response

A secondary local and splenic cell-mediated immune response was observed and compared to the primary response. Previous studies have demonstrated cell-mediated immunity (CMI) by lymphocytes from bronchopulmonary washings and have shown that its appearance is to a large extent inedpendent of splenic CMI. This study evaluated the secondary as compared to the primary response, with respect to both cellular and humoral immune responses. Guinea pigs were immunized with influenza virus vaccine either nasally or parenterally, booster immunizations were given by the same route, and animals were killed at various times after immunization or booster. The inhibition of macrophage migration was used to assess CMI. As in previous studies, local application of antigen led to mainly local appearance of CMI, whereas parenteral immunization led to mainly systemic CMI. Both pulmonary and splenic lymphocytes showed an inhibition of macrophage migration that appeared 2 to 3 days sooner after the booster, as compared to the primary immunization. There was no evidence, however, for the earlier production or increased amount of antibody in the bronchial secretions in the boosted animals. The results suggest that pulmonary as well as splenic T lymphocytes exhibit memory, but that pulmonary B lymphocytes do not.     

Immunity to human immunodeficiency virus (HIV) in children with chronic HIV infection receiving highly active antiretroviral therapy

Our objective was to describe the CD4-mediated human immunodeficiency virus (HIV)-specific cell-mediated immunity (CMI) and its virologic and immunologic correlates in children with chronic HIV infection on highly active antiretroviral therapy (HAART). Twelve HIV-infected children on stable antiretroviral therapy with a median level of CD4+ lymphocytes (CD4%) of 25.5% and a median viral load (VL) of 786 HIV RNA copies/ml were enrolled in this study. Nine of these children were also cytomegalovirus (CMV) seropositive. Blood mononuclear cells, stimulated with HIV and CMV antigens, were used to measure lymphocyte proliferation and to enumerate gamma interferon (IFN-gamma)-producing CD4+ cells. HIV CMI and CMV CMI were detected in similar proportions of patients and correlated with each other, although the HIV responses were less robust. HIV lymphocyte proliferation significantly increased with lower HIV VL and showed a trend to increase with higher CD4% and longer time on HAART. The in vitro IFN-gamma response to HIV or CMV was not affected by CD4%, VL, or HAART. Pediatric patients with established HIV infection on HAART frequently exhibit HIV CMI despite undetectable HIV replication. We concluded that the association between HIV CMI and CMV CMI indicates that the same factors govern responsiveness to either antigen.     

Immunity to Human Immunodeficiency Virus (HIV) in Children with Chronic HIV Infection Receiving Highly Active Antiretroviral Therapy

Our objective was to describe the CD4-mediated human immunodeficiency virus (HIV)-specific cell-mediated immunity (CMI) and its virologic and immunologic correlates in children with chronic HIV infection on highly active antiretroviral therapy (HAART). Twelve HIV-infected children on stable antiretroviral therapy with a median level of CD4⁺ lymphocytes (CD4%) of 25.5% and a median viral load (VL) of 786 HIV RNA copies/ml were enrolled in this study. Nine of these children were also cytomegalovirus (CMV) seropositive. Blood mononuclear cells, stimulated with HIV and CMV antigens, were used to measure lymphocyte proliferation and to enumerate gamma interferon (IFN-γ)-producing CD4⁺ cells. HIV CMI and CMV CMI were detected in similar proportions of patients and correlated with each other, although the HIV responses were less robust. HIV lymphocyte proliferation significantly increased with lower HIV VL and showed a trend to increase with higher CD4% and longer time on HAART. The in vitro IFN-γ response to HIV or CMV was not affected by CD4%, VL, or HAART. Pediatric patients with established HIV infection on HAART frequently exhibit HIV CMI despite undetectable HIV replication. We concluded that the association between HIV CMI and CMV CMI indicates that the same factors govern responsiveness to either antigen.     

Expression of cell surface antigens during allogeneic response studies with monoclonal antibodies to a polymorphic marker of activated lymphocytes and HLA class I and class II antigens

The kinetics of expression of cell surface antigens were studied during allogeneic stimulation and compared to 3H-thymidine incorporation. Two markers of T cell activation identified by monoclonal antibodies (monomorphic HLA-DR and a polymorphic blastic antigen) were compared to an ubiquitous antigen (beta 2m). beta 2m (Class I) and DR (Class II) antigens showed enhanced expression on alloactivated lymphocytes while the blastic antigen was only detected on alloactivated lymphocytes from serologically positive donors. The increased expression of these antigens could distinguish positive and negative mixed lymphocyte reactions and they preceded the rise in 3H-thymidine uptake.     

Cellular immune responses in asymptomatic human immunodeficiency virus type 1 (HIV-1) infection and effects of vaccination with recombinant envelope glycoprotein of HIV-1

Effects of human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein vaccines on cell-mediated immune (CMI) responses were assessed in HIV-1-infected patients. Asymptomatic, antiretroviral-treatment-na?ve, HIV-1-infected patients with CD4(+) T-cell counts greater than 400/microl received multiple intramuscular injections of HIV-1 IIIB recombinant envelope glycoprotein (rgp160) vaccine or HIV-1 MN recombinant envelope glycoprotein (rgp120) vaccine (eight patients, referred to as the HIV-1 vaccinees) or placebo or hepatitis B vaccine (three patients, referred to as the controls). Lymphocyte proliferation in response to HIV-1 envelope glycoproteins, both homologous and heterologous to the HIV-1 immunogens, was absent prior to study treatment in all patients but increased significantly during the vaccination series and after the final vaccination in HIV-1 vaccinees (P < 0.05) and remained absent in control patients. In flow cytometric analyses of intracellular cytokines, T-cell receptor stimulation with an anti-CD3 antibody induced gamma interferon (IFN-gamma) expression by activated CD4(+) and CD8(+) lymphocytes at greater frequencies than did stimulation with recombinant envelope glycoprotein and p24 of HIV-1 (P < 0.05). Mean frequencies of HIV-1 envelope glycoprotein-stimulated, activated intra-cellular IFN-gamma-producing CD4(+) and CD8(+) lymphocytes and of interleukin-2-producing CD4(+) lymphocytes did not increase after vaccination, but cytokine-producing cells were detectable in some patients. Comparing pre- to post-HIV-1 vaccination time points, changes in frequencies of activated, IFN-gamma-producing CD4(+) cells correlated inversely with changes in lymphocyte proliferation in response to recombinant envelope glycoprotein in HIV-1 vaccinees (P < 0.05). Increased CMI responses to HIV-1 envelope glycoprotein measured by lymphocyte proliferation were associated with HIV-1 recombinant envelope glycoprotein vaccines.     

Proliferative responses and gamma interferon and tumor necrosis factor production by lymphocytes isolated from tracheobroncheal lymph nodes and spleen of mice aerosol infected with Bordetella pertussis.

A group of mice was aerosol infected with live, virulent Bordetella pertussis bacteria. During a period of 7 weeks following the infection, with intervals of 1 week, lymphocytes were isolated from the tracheobroncheal lymph nodes (TBL) and the spleens (SPL) of the infected mice. The in vitro proliferative responses as well as the gamma interferon and tumor necrosis factor production levels of the isolated lymphocytes in response to stimulation with whole killed B. pertussis bacteria were measured as parameters for cell-mediated immunity (CMI). The course of the infection was monitored by counting of CFU in the lungs of the mice. Moreover, antibody responses in serum against a range of B. pertussis antigens were assessed. The results showed that a vigorous proliferative response of the TBL and SPL to stimulation with whole killed B. pertussis bacteria was induced by the infection. The proliferative response of the TBL was significantly higher than the response of the SPL. The proliferative responses were maximal 3 to 4 weeks after the infection and were paralleled by in vitro gamma interferon and tumor necrosis factor production upon specific stimulation. The development of the CMI was observed simultaneously with the clearance of the infection from the lungs. Antibody responses became measurable in the sera only after the infection was cleared. A specific CMI against pertussis toxin, the filamentous hemagglutinin, the 69-kDa outer membrane protein, and the agglutinogens 2 and 3, antigens which are under consideration for inclusion in future acellular pertussis vaccines, was successfully demonstrated in mice 3 weeks after the infection.     

Regulation of lymphocyte proliferation after influenza virus infection of human mononuclear leukocytes

Previous studies demonstrated that in vitro infection with influenza A viruses altered several functions of human monocytes-macrophages but did not detectably alter functions of human lymphocytes. However, both types of cells were infected, as determined by production and surface expression of viral antigens. In the current studies, human mononuclear leukocytes were infected in vitro and assayed for both influenza virus-induced proliferation and mitogen (phytohemagglutinin [PHAl)-induced proliferation, as well as for ability to stimulate proliferative responses by normal autologous leukocytes. The leukocytes showed proliferation in response to the infectious virus, but concomitant depressed proliferative responses to PHA. Coculture experiments suggested suppression of PHA-induced responses by the virus-infected cells. However, upon coculture with fresh autologous leukocytes (without PHA stimulation), both virus-infected macrophages and virus-infected lymphocytes induced autologous lymphocyte proliferative responses. Altered proliferative responses to mitogen stimulation after exposure to the virus were not due to diminished interleukin-1 production or diminished expression of HLA-DR by monocytes-macrophages. The expression of influenza virus antigens and resulting induction of autologous proliferative responses, combined with depressed mitogen-induced proliferation, may be important in human antiviral defense.     

The nature of the effector cells of cell-mediated immune responses to Sendai and Kunz virus infections in mice.

The development of virus-specific cell-mediated immune (CMI) responses to Sendai and Kunz virus infections of the respiratory tract of mice is described. In addition a heterotypic cell-mediated immune response develops during each of the virus infections. Cell depletion studies indicate that the effector cells of the virus-specific CMI responses are theta antigen bearing T lymphocytes. Similar studies of the heterotypic responses suggest that 2 cell types are involved, T lymphocytes and NK cells.     

Cell-mediated immune response of human lymphocytes to influenza A/USSR (H1N1) virus infection.

Cell-mediated immunity to influenza A/USSR (H1N1) virus was assessed by measuring transformation response and interferon production by Ficoll-Hypaque-purified peripheral blood lymphocytes from children and adults. Lymphocyte transformation was found to be related to the individual's previous experience with H1N1 influenza virus. Lymphocyte cultures, obtained from adults who had their last contact with the H1N1 virus over 20 years ago, were able to transform when incubated with H1N1 influenza antigens. This response suggests that influenza virus can induced long-term cell-mediated immunity for periods of at least 20 years. Interferon produced by stimulated lymphocytes was found to be unrelated to previous contact with influenza H1N1 virus; seronegative as well as seropositive individuals were capable of producing interferon in response to viral antigens. The interferon produced was type I.     

Comparison of different tests to measure immune responses to primate retroviruses.

The cell-mediated immune response to retrovirus antigens related to Mason-Pfizer virus (M-P V) and to baboon endogenous virus (BeV) was studied by lymphocyte transformation in blood samples from 90 women. Mitomycin C-treated infected cells and killed purified virions were used as antigens and were added on one or two occasions during in vitro stimulation. The number of activated lymphocytes was estimated by thymidine incorporation and by a virus plaque assay. The latter detected more responses to BeV antigens than the former, while the reverse was true with M-P V antigens. Assaying lymphocyte activity after one and two in vitro stimulations detected more responses than either test alone, but a high stimulation index after a first antigen addition was often followed by absence of response to a second antigen stimulus, indicating a possible in vitro suppressor mechanism. Human lymphocytes as well as those from experimentally infected monkeys responded to M-P V antigens present in infected cells but not to purified virions, while responses to the latter were found only in rabbits inoculated with the virus but not productively infected. Humoral antibodies are appeared in inoculated animals, as detected by 3 tests. Neutralization of syncytium forming units of M-P V showed sensitivity similar to an enzyme-linked immunosorbent assay and a radioimmunoassay but with both the latter specific results were obtained only when human and animal sera were preadsorbed with cell and calf serum antigens.     

The absence of differences in cellular immunity in elderly individuals with and without delayed local cutaneous reactions to influenza vaccine.

Two groups of elderly subjects who received bivalent influenza virus vaccine were identified. Those in Group I manifested marked local reactions, suggestive of delayed hypersensitivity reactions, whereas those in Group II, matched with Group I for vaccine preparation, had no cutaneous reactions. In vitro assays of immune function were performed on pairs of subjects from the two groups and included serum immunoglobulin and complement levels, antibody response to vaccine, lymphocyte transformation to mitogens (phytohemagglutinin, pokeweed mitogen and concanavalin A) and antigens (streptokinase-streptodornase, influenza virus vaccine antigens and multiple mixed lymphocytes), and polymorphonuclear leukocyte chemotactic responsiveness. There was no demonstrable correlation between delayed local cutaneous reaction and preservation of in vitro cell-mediated immune function in these elderly individuals.     

Stimulation of human lymphocyte subpopulations by protein A from Staphylococcus aureus

Protein A from Staphylococcus aureus is known to stimulate human lymphocytes. Using 3H-thymidine incorporation, virus plaque assay and induction of cytotoxic T lymphocytes (CTL), this study showed that soluble or insoluble protein A stimulated different lymphocyte subpopulations. Soluble protein A is highly mitogenic to T lymphocytes. In both 3H-thymidine incorporation and virus plaque assay, its maximum stimulation was as high as the stimulation by the nonspecific mitogens phytohemagglutinin and concanavalin A, and a higher CTL response than that induced by phytohemagglutinin or concanavalin A was induced. Mitogenic activity to B lymphocytes was negligible. S. aureus (Cowan I strain) is itself considered to be an insoluble form of protein A and is 3--4 times more mitogenic to B lymphocytes than pokeweed mitogen without any increase in virus plaque-forming cells. No mitogenicity was noted to T lymphocytes. Sepharose CL-4B-protein A, also known as insoluble protein A, stimulated both T and B lymphocytes effectively, but its mitogenicity to T lymphocytes was considered to be due to the soluble protein A released from the Sepharose CL-4B beads.     

In vitro stimulation of hamster lymphocytes with concanavalin A

The in vitro response of hamster lymphocytes to concanavalin A (Con A) was investigated by using the (3)H-thymidine incorporation assay, and the results obtained were compared to those with phytohemagglutinin (PHA-P). The optimum culture conditions for stimulation were obtained when one million lymph node cells were cultivated for 72 hr in 2 ml of RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum in the presence of either 2 mug of Con A or 0.05 ml of PHA-P. The average ratios of stimulation using lymph node cells with Con A and PHA-P were 227 and 14, respectively. Bone marrow and thymus cells responded very poorly or not at all. Methyl alpha-d-glucopyranoside, when added to the culture medium at the time of addition of Con A, completely inhibited stimulation. The lymphocyte stimulation could be reversed by addition of the sugar as late as 18 hr after the addition of Con A. The lymphocyte response from hamsters bearing tumors induced by simian virus 40 tumor cells and from hamsters immune to tumor transplants was comparable to the response of lymphocytes from healthy donors.     

Cell-mediated immunity in rubella assayed by cytotoxicity of supernatants from rubella virus-stimulated human lymphocyte cultures.

Rubella virus-stimulated lymphocytes from rubella-seropositive donors produced in the culture medium cytotoxic activity with preferential action against rubella-infected over uninfected target cells. The ability of lymphocytes to produce the cytotoxic activity upon stimulation by rubella virus correlated with the humoral rubella-immunity status, i.e. no such cytotoxic activity developed in the supernatants of lymphocyte cultures of rubella-seronegative donors. Stimulation of lymphocytes from seropositive donors by rubella virus was also detected by thymidine incorporation, but the correlation of lymphocyte responsiveness to the humoral rubella antibody status was not so clear as in the cytotoxicity assay. Conversion of lymphocytes from unresponsive to responsive to rubella virus following natural rubella infection and after rubella vaccination was demonstrated using both methods. Following vaccination rubella-specific cell-mediated immunity first became demonstrable at 14 days. The responsiveness of lymphocytes to phytohaemagglutinin (PHA) after rubella vaccination was followed by studying thymidine uptake and the ability of lymphocytes to produce lymphootoxin. By both tests marked suppression of PHA response occurred at days 3 and 7 after vaccination.