NK cell receptor/H2-Dk-dependent host resistance to viral infection is quantitatively modulated by H2q inhibitory signals

The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2(k), we generated double congenic mice between MA/My and BALB.K mice and an F(2) cross between FVB/N (H-2(q)) and BALB.K (H2(k)) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2(k) in conjunction with Cmv3(MA/My) or Cmv3(FVB) were resistant to MCMV infection. Subsequently, an F(3) cross was carried out between transgenic FVB/H2-D(k) and MHC-I deficient mice in which only the progeny expressing Cmv3(FVB) and a single H2-D(k) class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell-dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2(q) alleles influence the expression level of H2(q) molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2(q) alleles. Our results support a model in which H-2(q) molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-D(k) on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell-mediated control of viral load.     

An X-encoded alloantigenicity between BALB/c and C57BL/6 strains of mice

To detect minor barriers to histocompatibility that might be encoded on the X chromosome in mice, we grafted reciprocal sets of (C57BL/6xBALB/c)F1, (C57BL/6xDBA/2)F1, and (BALB/cxDBA/2)F1 mice with tail skin from the respective paternal inbred strain. Our histogenic analysis suggests that, compared with the C57BL/6 mouse strain, the BALB/c strain generates X-linked antigen loss. In contrast, we detected no X-linked histogenic differences between strains C57BL/6 and DBA/2, or DBA/2 and BALB/c. To localize this X-linked barrier to histocompatibility, we produced a panel of 25 [(BALB/cxC57BL/6)F1xC57BL/6]N2 males that were grafted with C57BL/6 skin to determine which carried the BALB/c-derived component(s) necessary for graft rejection. DNA marker analysis showed one region of overlapping BALB/c-derived X-chromosomal segments among the graft rejecters, suggesting that this antigen-loss haplotype ( H-hix(c), for histoincompatibility on the X chromosome, c haplotype) may be restricted within the DXMit55 to the Xq telomere interval (which excludes only the centromeric tip of the X). Further backcrossing of H-hix(c) to C57BL/6 resulted in fewer rejecter mice than expected by the N4 generation, suggesting that a second, unlinked locus is also involved in this X-linked alloantigenicity. The vigorous rejection of male (C57BL/6xBALB)F1 and female (B6.C- H2(d)xC57BL/6)F1 skin by (BALB/cxC57BL/6)F1 males, as well as the assessment of markers on Chromosome 17 among N2 and N4 graft-recipient males, suggests that this second locus is H2, and that H-hix(b)-encoded alloantigens require both H2(b) and H2(d)-encoded presentation molecules for efficient graft rejection.     

Antigen-binding receptors on T cells from long-term MLR. Evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (V_H) determinants strongly inhibited vesicle binding by both primary and longterm MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesiclebinding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectively blocked with the anti-V_H reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.Abbreviations used in this paper B10 C57BL/10 - Con A concanavalin A - FcR Fc receptor - FCS fetal calf serum - H heavy chain - Ia I-region associated antigen - Ig immunoglobulin - LPS lipopolysaccharide - Lyt T-lymphocyte differentiation antigen - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - PM plasma membrane - T thymus derived - Tcr T-cell receptor - V variable region of Ig     

The MHC locus controls size variations in the CD4 compartment of the mouse thymus

The genetic factors that determine the size of lymphocyte populations are largely unknown. We studied the genetic control of variations in the size of the CD4 thymic compartment in unmanipulated mouse strains. A time-course experiment showed that the C57BL/6 mouse strain has a consistently reduced proportion of thymic CD4 cells compared to the BALB/c strain. This difference denotes a decrease in the efficiency of the transition from CD4(+)CD8(+) to CD4(+) thymocytes in the C57BL/6 mouse. Genome-wide genetic mapping identified a major quantitative trait locus in the MHC region that controlled the variations in the proportion of CD4 thymocytes in an F2 intercrossed progeny from C57BL/6 and BALB/c mice (LOD score=21.7). The linkage was maximal at the MHC class II molecule Ea locus, which explained 59% of the observed phenotypic variance. As the C57BL/6 mouse does not express Ea, we hypothesize that the decreased size of the CD4 compartment in the C57BL/6 thymus is due to a reduction in the number of functional MHC class II genes. This study suggests that, at the level of the thymus, the MHC molecules in addition to conferring functional restriction and self-tolerance on the T-cell repertoire, also play a role in determining the homeostasis of the thymic compartments.     

Characterization of SJL T cell clones responsive to syngeneic lymphoma (RCS): RCS-specific clones are stimulated by activated B cells

To gain insight into the nature of the syngeneic T cell-stimulating molecules on SJL lymphoma cells (RCS), a panel of eight Ly-1+2- T cell clones that are specific for transplantable RCS has been generated. All of these clones proliferate vigorously in response to two independent RCS lines and to LPS-activated syngeneic or F1 B cell blasts, but not to unstimulated SJL spleen cells or to allogeneic B cell blasts. Only one RCS-specific clone displays a proliferative response to (SJL X BALB/c) resting spleen cells, suggesting that I-E molecules are not the source of stimulation of RCS-responsive cells. Responses of the T cell clones to both RCS and syngeneic LPS-activated B cells are inhibited by monoclonal antibodies to I-A antigens, and not by antibody to I-E antigens. These findings suggest that RCS-responsive T cells are stimulated either by syngeneic I-As alone, in a form expressed on activated B cells, or by I-As in combination with X, where X is a cell surface antigen present on B cells at certain stages of differentiation.     

Mechanisms of killing of measles virus-infected cells by human lymphocytes: interferon associated and unassociated cell-mediated cytotoxicity

The recent interest in natural killer (NK) cells in immunosurveillance and the ability of infection with certain organisms to modulate NK activity led us to examine the influence of Toxoplasma gondii infection on mouse NK cells. Infection of BALB/c mice with 5 × 10³ virulent Toxoplasma intraperitoneally (ip) resulted in significantly enhanced NK activity in peritoneal exudate cells (PC) and in spleen cells (SC). Intravenous (iv) and subcutaneous (sc) challenge of BALB/c mice with Toxoplasma also resulted in enhanced natural killer (NK) activity in PC and SC. In BALB/c mice, as well as in other strains (A/J, C57BL/6, C3H/HeJ, CeH/HeN, [A/J × C3H]F₁), peak augmentation of PC and SC NK activity was observed 3 days following ip Toxoplasma challenge. Administration of silica to mice abolished Toxoplasma-induced NK cytotoxicity. BALB/c mice chronically infected with Toxoplasma had significantly higher endogenous NK activity than did controls in PC but not in SC. Chronically infected BALB/c mice boosted with virulent Toxoplasma ip exhibited significantly enhanced NK activity in PC but not in SC. Thus, acute and chronic infection with Toxoplasma modulates NK activity in addition to macrophage activation and thereby provides a system that should facilitate study of the relative contribution of NK cells and activated macrophages in resistance to tumor growth and spread.     

Syngeneic responses by murine thymocytes: a role for non-MHC and non-MLS genes

A subpopulation of thymocytes from adult mice that is nonadherent to macrophage monolayers showed dramatically increased syngeneic mixed leukocyte responses (SMLR). Cloned cells were derived by limiting dilution from these SMLR-primed BALB/c thymocytes and were maintained and subcloned by repeated stimulation with syngeneic BALB/c spleen cells without the addition of exogenous interleukins. The cloned thymocytes were tested for their reactivities against H-2- and Mls-identical BALB/c and B10.D2 spleen cells (H-2d, Mlsb). We found that BALB/c and B10.D2 stimulator cells differed significantly in their capacity to restimulate the cloned BALB/c thymocytes. In addition, polyclonal syngeneic mixed leukocyte cultures (SMLC) of BALB/c thymocytes also showed differential restimulation by BALB/c and B10.D2 stimulators. Taken together, our data indicate a role for the product(s) specified by non-MHC and non-Mls gene(s) in the autorecognition by thymocytes.     

Abortive versus productive viral infection of dendritic cells with a paramyxovirus results in differential upregulation of select costimulatory molecules

Dendritic cells are the most potent antigen-presenting cell for priming naive T cells. Optimal activation of T cells requires that dendritic cells undergo a process of maturation resulting in the increased expression of costimulatory molecules, such as CD40, CD86, and CD80, and the production of cytokines. In this study we analyzed the effect of infection of dendritic cells obtained from two strains of mice, BALB/c and C57BL/6, with the paramyxovirus simian virus 5 (SV5). Our results show that C57BL/6 bone marrow-derived dendritic cells (BMDC) are much more permissive to infection with SV5 at a multiplicity of infection (MOI) of 10 PFU/cell compared to BALB/c BMDC, as determined by the production of viral proteins and progeny. However, infection of BALB/c BMDC with a higher MOI of 50 PFU/cell resulted in a productive infection with the production of significant amounts of viral proteins and progeny. Regardless of the permissivity to infection, both BALB/c and C57BL/6 BMDC efficiently upregulated CD40 and CD86. However, CD80 upregulation correlated with the level of expression of viral proteins and the production of viral progeny. While secreted alpha/beta interferon was required for increased expression of all three molecules, optimal CD80 expression was dependent on an additional signal provided by a productive viral infection. These findings provide evidence that the signals controlling the expression of costimulatory molecules following viral infection are distinct. Further, they suggest that the amount of virus encountered and/or the permissivity of a dendritic cell to infection can alter the resulting maturation phenotype and functional capacity of the infected dendritic cell.     

TheCmv1Host Resistance Locus Is Closely Linked to theLy49Multigene Family within the Natural Killer Cell Gene Complex on Mouse Chromosome 6

Natural killer (NK) cells play important roles in controlling tumor cells and against a range of infectious organisms. Recent studies of mouse NK cell surface receptors, which may be involved in the specificity of NK cells, have shown that many of these molecules are encoded by theLy49andLy55(Nkrp1) multigene families that map to distal mouse chromosome 6. Also mapping to this NK cell gene complex (NKC) is the resistance locus,Cmv1,which is involved in genetically determined resistance to murine cytomegalovirus (MCMV). The aim of this study was to localizeCmv1more precisely in relation to other NKC loci by generating a high-resolution genetic map of the region. We have analyzed 1250 backcross mice comprising panels of 700 (BALB/c × C57BL/6J)F₁× BALB/c and 550 (A/J × C57BL/6J)F₁× A/J progeny. A total of 25 polymorphic genes or microsatellite markers were analyzed over a region of 10 map units fromD6Mit134toD6Mit59.TheCmv1phenotypes of mice recombinant in this interval were tested by infection with MCMV. The results obtained indicate that the functionally important NKC region is a tightly linked cluster of loci spanning at least 0.4 map units. Furthermore,Cmv1maps distal to, but very closely linked to, theLy49multigene family (<0.2 map units), suggesting that MCMV resistance may be conferred by MHC class I-specific NK cell receptors.     

Degree of proliferative response to concanavalin A of spleen cells of mice of different strains and production of interleukin-2

Differences in the lymphoproliferative response to Con A of spleen cells allowed one to distinguish a high responder (BALB/c and DBA/2) and low responder (C57BL/6 and CC57BR) mice. BALB/c and DBA/2 mice (H-2d haplotype) produced interleukin 2 better, than C57BL/6 and CC57BR mice (H-2b haplotype). However acceptance of interleukin 2 was better in BALB/c and C57BL/6, than in DBA/2 and CC57BR mice. Summarizing these facts the authors suppose that the differences in interleukin 2 production and acceptance play an important role in the height of lymphoproliferative response.     

Strain difference in mouse natural killer activity augmented by mouse interferon and poly I.C

The level of natural killer (NK) activity was found to vary considerably among several mouse strains. In vivo and in vitro, interferon (IFN) and IFN inducers have been shown to augment mouse NK activity. C3H/He mice showed high NK activity, DDD/1 and A/J mice low NK activity, and C57BL/6, BALB/c and DBA/2 mice intermediate NK activity after injection with polyinosinic polycytidylic acid (poly I. C.). The same NK activity correlation was observed in nontreated mice, but the NK activities were lower compared with the poly I. C.-injected mice. Moreover, the DDD/1 and A/J mice showed almost no augmentation of NK activity on injection with poly I.C. In vivo, C3H/He, BALB/c and C57BL/6 mice injected with IFN showed augmented NK activity, but DDD/1 mice showed no such reaction. In vitro, C3H/He, BALB/c and C57BL/6 mouse spleen cells treated with IFN also showed augmented NK activity, but DDD/1 mouse spleen cells showed almost none. F1 hybrids between high (C3H/He) and low (DDD/1) NK-activity strains showed high NK activity. Thus, activity is dominant over low activity. The segregation of (DDD/1 X C3H/He) Fl X DDD/1 back-cross mice suggested that the strain differences in NK activity are under polygenic control.     

Exogenous interleukin-2 abrogates differences in the proliferative responses to T cell mitogens among inbred strains of mice.

The proliferative response of spleen cells from BALB/c mice to stimulation with a T cell mitogen, concanavalin A (Con A), was two or more times stronger than that of cells from C57BL/10SnSc (B10) mice. In contrast, the cells from B10 mice responded better to B cell mitogen bacterial lipopolysaccharide (LPS). The differences in the proliferative response to Con A stimulation were not associated with the function of macrophages nor did they depend on IL-1. Spleen cells from BALB/c and B10 mice synthesized comparable amounts of mRNA for IL-1 alpha, and the production of biologically active IL-1 was even higher in the B10 strain. Indomethacin, an inhibitor of prostaglandin synthesis, had no effect on the differences in reactivity between the cells from BALB/c and B10 mice. In addition, no differences in the synthesis of mRNA for the inducible 55-kDa interleukin-2 (IL-2) receptors were found between the spleen cells from BALB/c and B10 mice. However, Con A-stimulated spleen cells from B10 mice produced a significantly lower amount of biologically active IL-2 than similarly stimulated cells from BALB/c mice. In the presence of exogenous IL-2, these low responder spleen cells from the B10 mice responded by proliferation to Con A stimulation to the same extent as cells from the BALB/c mice. These results thus show that a low proliferative response to Con A stimulation in B10 mice was a consequence of a lower production of IL-2 and possibly abrogated the proliferative hyporeactivity produced by exogenous IL-2. We suggest that the differences in the ability to produce IL-2 could be a reason for the discrepancies observed in the immunological responsiveness between BALB/c and B10 mice.     

Effect of class I MHC binding peptides on recognition by natural killer cells.

NK cells can recognize and destroy a broad range of cells, including many tumor cells and virally infected cells, yet spare most normal cells. Identification of the target structure recognized by these cells has proved elusive. An attractive hypothesis is that, unlike B cells and T cells that recognize a specific foreign marker, NK cells respond to the absence of a "self" marker. Class I MHC molecules have been implicated as the self markers whose absence can trigger lysis. We show here that normal cells are lysed on incubation with IL-2-activated NK cells if peptides that can bind to the class I MHC molecules of the normal cells are also included in the assay, and speculate that this binding is somehow removing a self marker that normally protects a cell from lysis. NK cells were derived from splenocytes of young (5 to 8 wk old) athymic nude BALB/c (H-2d) or nude C57Bl/6 (H-2b) mice incubated with 1000 U/ml rIL-2, and target cells were derived from splenocytes of normal BALB/c or C57Bl/6 mice incubated with Con A. Peptides were from xenogeneic, viral, self, and mutated self protein sequences and included sequences specific for Kd, Kb, Db, and Ld. All peptides increased lysability of those targets to which they could bind.     

The immunoglobulin <Emphasis Type="Italic">IGHD</Emphasis> gene locus in C57BL/6 mice

Four immunoglobulin heavy chain diversity (IGHD) gene subgroups (DFL16, DSP2, DQ52, and DST4) have been identified previously in BALB/c mice. Although the locations of most IGHD genes have been established based on restriction map and Southern blot analysis, a complete mouse IGHD gene locus map at the sequence level is still not available. In addition, a previous restriction fragment length polymorphism study suggested that significant difference in the IGHD gene locus exists between C57BL/6 and BALB/c mice. The author has now analyzed the C57BL/6 mouse genomic data and established a complete map of the IGHD gene locus. All four IGHD subgroups previously identified in BALB/c mice were found to be present in C57BL/6 mice. However, unlike the BALB/c mice, which have at least 13 IGHD genes, the C57BL/6 genome contains only ten IGHD genes, which include one DFL16, six DSP2, one DQ52, and two DST4 genes. There are also differences in the coding regions of the DST4 and DQ52 genes between the two mouse strains.     

Dendritic cell-induced activation of adaptive and innate antitumor immunity

While studying Ag-pulsed syngeneic dendritic cell (DC) immunization, we discovered that surprisingly, unpulsed DCs induced protection against tumor lung metastases resulting from i.v. injection of a syngeneic BALB/c colon carcinoma CT26 or a syngeneic C57BL/6 lung carcinoma LL/2. Splenocytes or immature splenic DCs did not protect. The protection was mediated by NK cells, in that it was abrogated by treatment with anti-asialo-GM1 but not anti-CD8, and was induced by CD1(-/-) DCs unable to stimulate NKT cells, but did not occur in beige mice lacking NK cells. Protection correlated with increased NK activity, and increased infiltration of NK but not CD8(+) cells in lungs of tumor-bearing mice. Protection depended on the presence of costimulatory molecules CD80, CD86, and CD40 on the DCs, but surprisingly did not require DCs that could make IL-12 or IL-15. Unexpectedly, protection sensitive to anti-asialo-GM1 and increased NK activity were still present 14 mo after DC injection. As NK cells lack memory, we found by depletion that CD4(+) not CD8(+) T cells were required for induction of the NK antitumor response. The role of DCs and CD4(+) T cells provides a novel mechanism for NK cell induction and innate immunity against cancer that may have potential in preventing clinical metastases.     

Trypanosoma cruzi: the expansion of NK, T, and NKT cells in the experimental infection

T and NK cells play a key role in resistance to Trypanosoma cruzi infections, mainly through IFN-gamma production. The expression of T and NK cells surface markers was studied in NWNA spleen cells of resistant C3H and susceptible BALB/c mice that release IFN-gamma in the early and late acute infection, respectively. In the progressively enlarged spleens, we found: (a) an increased percentage and number of NK blast cells as early as at 2 days post-infection (pi), (b) an enrichment of T and NK cells, in both the total and blast populations, during the late acute phase. At 17 days pi, there was also an accumulation of TCR- alphabeta+DX5+, NKT cells, mainly in resistant mice. At 21 days pi, the enrichment of NK cells ceased, while spleen cells and the T cell compartment continued their expansion. In the chronic stage, TCR-alphabeta+ blasts were expanded in both mouse strains, but NK blasts increased only in BALB/c that, unlike C3H mice, release IFN-gamma. As T and NK cell proliferation is not always associated to IFN-gamma release the experimental downregulation of their expansion to avoid tissue damage could be explored.     

A locus on mouse chromosome 6 that determines resistance to herpes simplex virus also influences reactivation, while an unlinked locus augments resistance of female mice

During studies to determine a role for tumor necrosis factor (TNF) in herpes simplex virus type 1 (HSV-1) infection using TNF receptor null mutant mice, we discovered a genetic locus, closely linked to the TNF p55 receptor (Tnfrsf1a) gene on mouse chromosome 6 (c6), that determines resistance or susceptibility to HSV-1. We named this locus the herpes resistance locus, Hrl, and showed that it also mediates resistance to HSV-2. Hrl has at least two alleles, Hrl(r), expressed by resistant strains like C57BL/6 (B6), and Hrl(s), expressed by susceptible strains like 129S6 (129) and BALB/c. Although Hrl is inherited as an autosomal dominant gene, resistance to HSV-1 is strongly sex biased such that female mice are significantly more resistant than male mice. Analysis of backcrosses between resistant B6 and susceptible 129 mice revealed that a second locus, tentatively named the sex modifier locus, Sml, functions to augment resistance of female mice. Besides determining resistance, Hrl is one of several genes involved in the control of HSV-1 replication in the eye and ganglion. Remarkably, Hrl also affects reactivation of HSV-1, possibly by interaction with some unknown gene(s). We showed that Hrl is distinct from Cmv1, the gene that determines resistance to murine cytomegalovirus, which is encoded in the major NK cell complex just distal of p55 on c6. Hrl has been mapped to a roughly 5-centimorgan interval on c6, and current efforts are focused on obtaining a high-resolution map for Hrl.     

Anti-idiotypic antibodies against UV-induced tumor-specific CTL clones. Preparation in syngeneic combination

In this study, we first established several CTL clones of (BALB/c x C57BL/6)F1 origin that were specific for either syngeneic UV female 1 or UV male 1 fibrosarcoma cell lines. All the CTL clones had Thy-1+ Lyt-2+ L3T4- phenotypes and showed Kd restriction when lysing the corresponding target cells. Sera obtained from syngeneic animals immunized with three CTL clones, 10B-5 for UV female 1, and CTL9 and CTL10 for UV male 1, showed specific inhibition of target cell lysis with the corresponding CTL clones. The inhibitory activities were found in sera of the majority of immunized animals. Because the inhibitory activity resides in protein A-binding fraction, mAb were produced by hybridizing spleen cells of hyperimmune animals. N1-56 was thus obtained from a mouse immunized with 10B-5 CTL clone reactive with UV female 1. N1-56 was clonotype specific, reacting with 10B-5 but not with other CTL lines or leukemia cell lines. No N1-56+ cells were detectable in thymocytes, lymph node cells, or spleen cells of either naive or UV female 1-immune CB6F1 mice. Immunoprecipitation showed that N1-56 reacts with 90,000 Mr molecules on 10B-5 CTL clone under nonreducing conditions and 45,000 Mr molecules under reducing conditions, indicating its reactivities with idiotypic determinants of TCR on the CTL clone. N1-56 inhibited lytic activity of 10B-5, but neither N1-56 nor alpha-10B-5 hyperimmune serum inhibited that of alpha-UV female 1 mixed lymphocyte tumor cell culture cells. N1-56 induced proliferation of 10B-5 without addition of Ag.