Adoptive transfer of delayed-type hypersensitivity to Sendai virus. I. Induction of two different subsets of T lymphocytes which differ in H-2 restriction as well as in the Lyt phenotype

This paper investigates kinetics and antigenic and genetic requirements for transfer of delayed-type hypersensitivity (DTH) reactions against Sendai virus. Kinetics of sensitization of effector cells are similar to those described in other virus systems. Maximum DTH response is elicited in the footpad 7 days after sensitization; maximal increase in footpad thickness is found approximately 24 hr after injection of virus and transfer of immune lymphocytes. DTH effector cells are Ig^? and are susceptible to treatment with anti-theta antibody and complement. After transfer of immune lymphocytes a DTH reaction can be elicited by infectious virus, uv light-inactivated virus or cell-cultured Sendai virus which lacks fusion capacity. Requirements of H-2 compatibility are dependent on the biological nature of the virus preparation used for challenge: High doses of infectious or uv light-inactivated Sendai virus require K, D, or IA region compatibility; low concentrations of infectious virus require K and/or D region compatibility, while cell-cultured fusion-negative Sendai virus requires IA region homology. At the level of induction K, D region-restricted DTH-effector T lymphocytes (Td) and cytolytic T lymphocytes (Tc) are stimulated to a greater extent by infectious virus than by uv light-inactivated virus. Furthermore, stimulation of these T lymphocytes is dependent on the route chosen for immunization: intravenous (iv) injection is superior to intraperitoneal (ip) injection; subcutaneous (sc) injection causes the lowest degree of sensitization. IA region-restricted Td lymphocytes are stimulated to comparable amounts by infectious or uv light-inactivated Sendai virus independent of the route of immunization. Td lymphocytes, which require IA region compatibility and Td lymphocytes which require K or D region compatibility can be distinguished by their Lyt phenotype, e.g., IA region-restricted Td lymphocytes are characterized by Lyt-1⁺2^? antigens, while K or D region-restricted Td lymphocytes are phenotypically Lyt-1(⁺)2⁺.     

Macrophage procoagulant-inducing activity of influenza-specific effector T cells

Three different types of immune mouse T cells raised against influenza virus were tested for their ability to induce the formation of macrophage procoagulant activity (MPCA) by a macrophage cell line PU5-1.8. They were primary spleen cells, taken 6 days after iv injection of virus, spleen cells from sensitized mice challenged with virus and cultured in vitro for 5 days (secondary cultured cells), and cloned T cells. With the last two preparations, some samples were K,D region restricted, Lyt 2⁺, and had cytotoxic activity; other samples were I region restricted, Lyt 2^?, and were not cytotoxic. Samples of a concanavalin A-activated T-cell supernatant which regularly induced MPCA with PU5-1.8 cells were included as controls in all assays. A few batches of T-cell preparations failed to induce MPCA production, however, most batches were active. Two sources of variation were detected: first, the number of cells (5-to 150-fold) needed to induce a certain level of MPCA, as measured by the decrease in clotting time; and second, the value of the gradient of the cell dose response. Both K,D- and I-region-restricted cells, either as cloned or secondary cultured cells, could induce MPCA but with the latter preparation, I-region-restricted cells were the better inducers by about eightfold. T cells tested in this way were also injected into mouse hind footpads and their ability to mediate delayed-type hypersensitivity (DTH) reactions was measured. A positive but not proportional correlation between the abilities to induce MPCA and mediate DTH activity for primary spleen cells was found, but this was not generally observed with cultured or cloned T cells.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

Correlation of tumor specific delayed type hypersensitivity reaction and tumor protection to SV40-induced mKSA fibrosarcoma

Summary Mice immunized by excision of a primary, subcutaneously growing SV₄₀-induced mKSA solid tumor which resisted challenge of homologous tumor cells administered at a contralateral site, were found to develop a specific DTH response to SV₄₀ tumor associated transplantation antigens (TATA).In a two-way criss-cross experiment, this DTH response (assessed by direct challenge) was found to be one-way SV₄₀ specific in that chemically induced, non SV₄₀, MCA tumor failed to elicit a DTH response in mice primed by excision of mKSA tumor.These mice also showed a corresponding one-way specific protection against challenge with live homologous mKSA sarcoma cells. In contrast immunization and challenge of MCA-excised mice with either MCA or mKSA tumor cells, exhibited cross-reactivity in both DTH response and protection against either tumor.Unlike this cross-immunity by the direct challenge method, transfer of immune spleen cells from mKSA or MCA excision-primed mice demonstrated a specific DTH response and protection to the original immunizing, homologous but not heterologous tumor. Tumor resistant, DTH-primed mice remained DTH reactive to the primary tumor cells over a period of 4 weeks. Characterization of the splenic T-DTH cells in mice primed by excision of mKSA tumor, indicated a Lyt 1⁺2⁺ phenotype of cells conferring both the DTH response and the immune protection against mKSA sarcoma in a local (Winn) adoptive transfer assay, thus reinforcing the correlation between the DTH response and the antitumor protection.     

The Lyt phenotype of the T cells in an antitumor adoptive transfer differs for “parent to F1” and “parent to parent” combinations

The T-cell subset mediating tumor graft neutralization was characterized in a methylcholanthrene (MC) tumor system. Lyt 1⁺ cells were critical for the successful prevention of outgrowth of the tumor cells in graft neutralization assays with irradiated recipients. Elimination of Lyt 1⁺ cells abolished the outgrowth inhibitory effect exerted by T-cell-enriched populations derived from syngeneic or semisyngeneic mice immunized with the H-2-carrying MC-induced M-A tumor. In accordance, lymphocyte populations containing 98% Lyt 1⁺ cells derived from M-A-immune mice, mediated a complete transplantation immunity against this tumor. When the immune T cells admixed to the tumor inoculum were syngeneic to the recipient (i.e., A-derived cells were transferred to A recipients, or F₁ to F₁), elimination of the Lyt 2⁺ cells did not influence the potential to inhibit tumor outgrowth. The presence of Lyt 1⁺2^? cells were thus necessary, and sufficient, in the syngenic combination. A reduction of the graft-inhibiting potential occurred after elimination of Lyt 2⁺ cells from the A-derived M-A immune T-cell population when the recipients were semisyngeneic (i.e., (A/Sn X A.SW)F₁, (A/Sn X CBA)F₁, or (A/Sn X C57Bl/6)F₁). Consequently, only in the semisyngeneic, but not in the syngeneic combinations, was the transfer of Lyt 2⁺ cells necessary for optimal graft inhibition. It can be concluded that the genotypic relation between the donor and the recipient influences the prerequisites of the tumor cell neutralization.     

Eradication of a large MOPC-315 tumor in athymic nude mice by chemoimmunotherapy with Lyt2+ splenic T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor

Summary We have previously shown that spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose (2.5 mg/kg) melphalan (l-phenylalanine mustard) therapy are effective in preventing tumor progression upon adoptive transfer into BALB/c mice bearing a barely palpable tumor that had been treated with a subcurative dose of melphalan [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that such spleen cells in conjunction with a subcurative dose of drug (adoptive chemoimmunotherapy, ACIT) can cause the complete regression of a large (15–20 mm) s.c. MOPC-315 tumor in a large percentage of T-cell-deficient (athymic nude) tumor-bearing mice. Spleen cells that were effective in ACIT of athymic nude mice displayed in vitro a substantial direct lytic activity against MOPC-315 tumor cells, and the lytic activity was greatly enhanced when the spleen cells were cultured for 5 days with or without mitomycin-C-treated MOPC-315 stimulator tumor cells. The cells responsible for the therapeutic effectiveness of the spleen cells in ACIT of athymic nude mice, as well as the cells responsible for the direct in vitro anti-MOPC-315 lytic activity of the spleen cells, were of the Lyt 2 and not the L3T4 phenotype. Most of the athymic nude mice that completely eradicated a large MOPC-315 tumor as a consequence of ACIT were capable of rejecting a challenge with 30–100 times the minimal lethal tumor dose for 100% of normal BALB/c mice administered more than 1 month after the ACIT. The ability of these athymic nude mice to resist the tumor challenge was associated with the presence of a greatly elevated percentage of cells expressing T cell surface markers in their spleens. Thus, it is conceivable that splenic Lyt 2⁺ T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor mediate their therapeutic effectiveness in ACIT of athymic nude mice bearing a large MOPC-315 tumor, at least in part, through direct cytotoxicity for MOPC-315 tumor cells. In addition, eradication of a large MOPC-315 tumor through cooperation between antitumor immunity and melphalan toxicity endues the athymic nude mice with an elevated percentage of T cells in their secondary lymphoid organs, and these T cells are probably responsible for the long-lasting protective antitumor immunity exhibited by these mice.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01350 from the National Cancer Institute     

Transplantation tolerance: Evidence for Lyt 1+,2−,Qa 1.2+ suppressor-inducer T cells in allogeneic thymus-grafted nude mice

Nude mice, of BALB/c genotype, grafted with thymus stroma become immunocompetent (R. Hong, H. Schultz-Wisserman, E. Jarreth-Toth, S. D. Horowitz, and D. D. Manning, J. Exp. Med.149, 398, 1979; B. P. Chen and G. A. Splitter, Cell. Immunol.51, 127, 1980), but are tolerant to the thymus-donor genotype. Using such mice to investigate the mechanism(s) of transplantation tolerance, it was found that maintenance of tolerance required active interactions of three subsets of T cells specific for alloantigens of the thymus-donor genotype: (i) Lyt 1⁺,2^? helper T cells, (ii) Lyt 1^?,2⁺ suppressor T cells, and (iii) Lyt 1⁺,2^?,Qa 1.2⁺ suppressor-inducer T cells. In mixed-lymphocyte culture, helper T cells could be activated by alloantigens of the thymus-donor genotype, but clonal expansion of these helper T cells was inhibited by suppressor T cells with the same specificity. Furthermore, exogenous interleukin-2 (IL-2) could modulate this suppressor activity, which suggested that one consequence of suppression was to limit IL-2 available to effector T cells. The response of cultures to exogenous IL-2 also indicated that thymus alloantigen-specific helper T cells had functional IL-2 receptors. Last, the presence of Lyt 1⁺,2^?,Qa 1.2⁺ suppressor-inducer T cells were essential for active suppression, as suppressor T cells could not prevent helper T cells from proliferating to thymus-donor alloantigens when Lyt 1⁺,2^?,Qa 1.2⁺ cells were removed. Altogether, the data presented in this study indicate a feedback-suppression pathway that led to clonal silencing of effector cells in transplantation tolerance.     

Thymic lymphocytes: II. Phenotypic modifications of thymocytes after concanavalin A stimulation in the presence of interleukin 2: Early modifications of Lyt 1+2+ subset and later proliferation of cells with more mature phenotypes

Cortical thymocytes are devoid of any immune function, as tested by presently available techniques. The ability of this subpopulation to respond to mitogens or antigens in the presence of interleukin 2 (IL-2) produced by activated mature T lymphocytes has been claimed but is still questioned. In an attempt to study the participation of the different thymocyte subsets and especially that of the cortical type, phenotypic modifications were examined during concanavalin A activation in the presence of IL-2. An immunofluorescent double labeling technique with anti-Lyt 1 and anti-Lyt 2 antibodies was used which led to the determination of four different phenotypes: Lyt 1⁺2⁺, Lyt 1⁺2^?, Lyt 1^?2⁺, and Lyt 1^?2^?. Careful analysis of cell viability in culture and expression of the results in absolute numbers of living cells per culture allowed us to follow modifications of small cellular subsets. Cultures of total thymocytes and PNA-agglutinated (enriched in Lyt 1⁺2⁺ cells) and non-PNA-agglutinated cells (enriched in Lyt 1⁺2^?, Lyt 1^?2⁺, and Lyt 1^?2^? cells) were studied. It was shown that thymocyte activation began by early phenotypic modifications which took place within the first 2 hr of culture but only when Con A plus IL-2 were used. These modifications imply the reduction of the Lyt 1⁺2⁺ pool and a compensatory enhancement of Lyt 1^?2⁺ and Lyt 1^?2^? cells, without modification of the total cell number or [³H]thymidine incorporation. These early phenotypic changes are interpreted as the modulation of antigens on the surface of Lyt 1⁺2⁺ cells. The second phase of thymocyte activation implies cell death (essentially Lyt 1⁺2⁺ cells) and cell proliferation. The cells which specifically proliferate in the presence of Con A and IL-2 are Lyt 1⁺2^? and Lyt 1^?2⁺, the latter always being present in greater number. Cell survival and absolute number of Lyt 1⁺2^? and Lyt 1^?2⁺ cells in the activated PNA^?-enriched population are always higher than in total thymocyte and PNA⁺ cells cultures. Thus, if Lyt 1⁺2⁺ cortical thymocytes do not proliferate by themselves, they seem to intervene by providing Lyt 1^?2⁺ cells which proliferate secondarily.     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

Alveolar Macrophages Are Essential for Protection from Respiratory Failure and Associated Morbidity following Influenza Virus Infection

Alveolar macrophages (AM) are critical for defense against bacterial and fungal infections. However, a definitive role of AM in viral infections remains unclear. We here report that AM play a key role in survival to influenza and vaccinia virus infection by maintaining lung function and thereby protecting from asphyxiation. Absence of AM in GM-CSF-deficient (Csf2 ^−/−) mice or selective AM depletion in wild-type mice resulted in impaired gas exchange and fatal hypoxia associated with severe morbidity to influenza virus infection, while viral clearance was affected moderately. Virus-induced morbidity was far more severe in Csf2 ^−/− mice lacking AM, as compared to Batf3-deficient mice lacking CD8α⁺ and CD103⁺ DCs. Csf2 ^−/− mice showed intact anti-viral CD8⁺ T cell responses despite slightly impaired CD103⁺ DC development. Importantly, selective reconstitution of AM development in Csf2rb ^−/− mice by neonatal transfer of wild-type AM progenitors prevented severe morbidity and mortality, demonstrating that absence of AM alone is responsible for disease severity in mice lacking GM-CSF or its receptor. In addition, CD11c-Cre/Pparg ^fl/fl mice with a defect in AM but normal adaptive immunity showed increased morbidity and lung failure to influenza virus. Taken together, our results suggest a superior role of AM compared to CD103⁺ DCs in protection from acute influenza and vaccinia virus infection-induced morbidity and mortality.     

Expression of T-cell differentiation antigens on activated Thymocytes

The expression of Thy 1, TL, Lyt1, and Lyt2 antigens on resting and proliferating thymocytes activated by concanavalin A in the presence of interleukin 2 has been studied by conventional complement-dependent cytotoxicity assay. The predominant population of resting thymocytes has a TL⁺Lytl⁺Lyt2⁺ phenotype while the predominant population of proliferating thymocytes has a TL — Lytl⁺Lyt2⁺ phenotype. Using several separation procedures such as agglutination by peanut lectin, BSA density gradient centrifugation, and pretreatment with high dilutions of anti-H-2 serum it was impossible to obtain a 100% pure population of TL⁺Lytl⁺Lyt2⁺ cells, suggesting that the population of resting immature thymocytes contains small subpopulations of phenotypically differentiated cells. The population of proliferating thymocytes is also phenotypically heterogenous and contains cells bearing all phenotypes that were described for different stages of T-cell differentiation, including TL⁺Lytl⁺Lyt2^? and TL⁺Lytl^?Lyt2⁺ with the following approximate frequency: TL⁺Lytl⁺Lyt2⁺—27%, TL⁺Lytl⁺Lyt2^?—8%, TL⁺Lyt1^?Lyt2⁺—4%, TL^?Lytl⁺Lyt2⁺—45%, TL^?Lyt1⁺Lyt2^?—13%, TL^?Lyt1^?Lyt2⁺—3%.     

Protection of mice against influenza virus infection: Enhancement of nonspecific cellular responses by Corynebacterium parvum

Groups of C57Bl/6J, BALB/c, BALB/c nu⁺/nu⁺ mice, inoculated intranasally with Corynebacterium parvum (350 μg/mouse) were protected from death by an otherwise lethal dose of influenza virus, A/WSN (H1N1) inoculated 3 days later. The lungs of C. parvum-trented, virus-infected C57Bl/6J, BALB/c, or BALB/c nu⁺/nu⁺ mice contained significantly less infectious virus than did controls, and this reduction was apparent as soon as 24 hr after virus inoculation. The maximum protective effect correlated with increased lung interferon levels. C. parvum treatment caused an increase in the lung cell number which was in part due to a large increase (ca. 10-fold) in macrophage content, and the natural killer cell activity was also enhanced, though not as markedly as occurred 3 days after infection. Most (>85%) of the resident macrophages in normal lungs were susceptible to infection by virus (as indicated by hemadsorption), whereas most of those recovered from the lungs of C. parvum-treated mice resisted infection. Despite the increase in macrophage content, the level of specific immune responses to infection, such as cytotoxic T-cell activity, DTH reaction, and antihemagglutinin antibody, remained unchanged by C. parvum treatment so that the major if not only effect of this treatment was on the level of the less-specific components of the immune system.     

Adoptive Transfer of EBV Specific CD8+ T Cell Clones Can Transiently Control EBV Infection in Humanized Mice

Epstein Barr virus (EBV) infection expands CD8⁺ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8⁺ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8⁺ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8⁺ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.     

Experimental influenza caused by an incomplete form of the agent]

Incomplete form of the influenza virus obtained in accordance with Nayak's method was administered intranasally to mice CBA and C57BL. From the lung tissue of the infected mice the causative agent could be isolated for 45 days, and from the other internal organs--the first hours after the infection only. In morphological investigation of the lungs of animals infected with an incomplete form of the influenza virus a prevalence of the proliferative component against the background of inflammatory changes was noted. Three months after the infection limited lymphoid formations consisting of monomorphic cells with hyperchromic nuclei were defined in the lung tissue. Marked proliferation of the alveolar and bronchial epithelium was observed later; considerable anaplasia of the cells was noted in the papillomatous structure of the alveolar and bronchial epithelium. Glomangioma of the mesentery was observed among affections of other internal organs in 18.7% of mice CBA.     

Delayed-type hypersensitivity responses to H-Y: Characterization and mapping ofIr genes

This paper examines the delayed-type hypersensitivity (DTH) response to male (H-Y) antigen(s). Female mice of theH?2 ^b haplotype developed delayed footpad reaction to syngeneic or allogenic male thymus and spleen cells after priming with syngeneic male thymus and spleen cells. The reaction peaks at 24 h, has classical DTH histology and is specific to H-Y antigen as it is not elicited with female cells. Cell transfer studies show that donor/recipient matching at theI?B ^b subregion is necessary for sucessful transfer of DTH and that the effective primed population is Thy-1⁺, Lyt-1⁺, 2^?. DTH response to H-Y antigen appears to be confined to mice of theH?2 ^b haplotype. There appears to be a lack of associative recognition between H-Y antigen and MHC-coded determinants in the effector phase of DTH, and macrophage processing of H-Y seems likely, since nonresponder haplotypes can elicit the DTH response. Studies withH?2 ^b recombinant mouse strains indicate that the dominantIr gene is located in theI?B region. Female F₁ hybrid mice derived from matings of strains not involvingH?2 ^b haplotype failed to develop DTH to H-Y. In summary, these data imply that a complete correlation exists between DTH to H-Y and the ability to reject male skin graft, suggesting that the effector mechanisms of skin-graft rejection may closely involve DTH cells.     

Tissue-Protective Effects of NKG2A in Immune-Mediated Clearance of Virus Infection

Virus infection triggers a CD8⁺ T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8⁺ T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1^b, which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8⁺ T cells. We have previously shown that Qa1^b-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8⁺ T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8⁺ effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8⁺ T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8⁺ T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8⁺ NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue integrity during virus clearance.     

Regulation by the H-2 gene complex of delayed type hypersensitivity

Identity at the major histocompatibility complex (MHC) was essential for successful transfer of delayed type hypersensitivity (DTH) in mice. The regions of the MHC involved differed according to the antigen used for sensitization. In the case of fowl gamma globulin (FGG), identity atI-A was necessary, whereas with dinitrofluorobenzene (DNFB), identity at theK, I, orD region was sufficient. These different genetic constraints probably reflect differences in the mechanisms by which antigens are presented to T lymphocytes. Cells from sensitized (CBA×C57BL)F₁ mice transferred DTH to FGG into parental-strain mice, but transfer was more effective in C57BL than in CBA with the same cell dose. This phenomenon is governed by the MHC, since there was better transfer intoH-2 ^b than intoH-2 ^k mice, regardless of their backgrounds. It may reflect the activity of an Ir gene-dependent process. Cells of one genotype (e.g., CBA), sensitized in chimeric mice derived from two MHC-incompatible strains (CBAC57BL), transferred DTH to both strains. These results do not support the notion that the genetic constraint observed in DTH transfer may be a result of the necessity for sensitized T and stimulator cells to match an identical MHC-coded cell interaction molecule. Rather, they favor the hypothesis that T cells recognize antigen, not as a naked determinant, but in close association with products of genes of the MHC.     

Impaired Clearance of Influenza A Virus in Obese,Leptin Receptor Deficient Mice Is Independent of Leptin Signaling in the Lung Epithelium and Macrophages

Rationale

During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients.

Methods

We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepR^fl/fl) or macrophages and alveolar type II cells (LysM-Cre/Lepr^fl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity.

Results

The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^fl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^{fl /fl} mice exhibited improved survival.

Conclusions

Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.     

In vivo effects of cyclosporine on influenza A virus-infected mice

Cyclosporine (cyclosporin A, CsA) administered to mice substantially affects their immune response to an influenza A virus infection. If treated with CsA for 21 days, the mouse lungs contain high titers of virus which are cleared more slowly than in controls. Indicators of pathological damage--lung weight, extent of consolidation, fine morphology, and the extent of infiltration of dividing cells into the lung--showed that administration of CsA greatly decreased the level of inflammation. The production of hemagglutination-inhibiting (HI) antibody was delayed but reached almost control levels and NK cell activity in the lung was also comparable to control levels. In contrast, a delayed-type hypersensitivity (DTH) response to the virus could not be elicited in the CsA-treated, infected mice at 6 or 12 days after infection. Cytotoxic-T-cell (Tc-cell) activity was present in the lungs of such mice though its appearance was delayed and the activity recovered was less than that of the control infected mice. If administered with a dose of virus lethal for normal mice. CSA-treated mice survived, probably due to the greatly reduced level of immunopathological damage in the infected lung.     

CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection

Liver macrophages internalize circulating bloodborne parasites. It remains poorly understood how this process affects the fate of the macrophages and T cell responses in the liver. Here, we report that infection by Trypanosoma brucei induced depletion of macrophages in the liver, leading to the repopulation of CXCL16-secreting intrahepatic macrophages, associated with substantial accumulation of CXCR6⁺CD4⁺ T cells in the liver. Interestingly, disruption of CXCR6 signaling did not affect control of the parasitemia, but significantly enhanced the survival of infected mice, associated with reduced inflammation and liver injury. Infected CXCR6 deficient mice displayed a reduced accumulation of CD4⁺ T cells in the liver; adoptive transfer experiments suggested that the reduction of CD4⁺ T cells in the liver was attributed to a cell intrinsic property of CXCR6 deficient CD4⁺ T cells. Importantly, infected CXCR6 deficient mice receiving wild-type CD4⁺ T cells survived significantly shorter than those receiving CXCR6 deficient CD4⁺ T cells, demonstrating that CXCR6⁺CD4⁺ T cells promote the mortality. We conclude that infection of T. brucei leads to depletion and repopulation of liver macrophages, associated with a substantial influx of CXCR6⁺CD4⁺ T cells that mediates mortality.