The genetic control of natural killer cell activity and its association with in vivo resistance against a moloney lymphoma isograft

Spleens of normal young mice of certain strains contain lymphocytes that can kill strain A-derived YAC-1 lymphoma cells in a⁵¹Cr release cytotoxic assay in vitro. We have previously classified mouse genotypes as high or low reactors, according to their responses in this test. In vivo resistance to small numbers of YAC ascites lymphoma cells is correlated with in vitro cytolytic activity. In vitro and in vivo tests were carried out on the same individual (A x C57BL)F₁ x A backcross mice. Natural in vitro killer cell activity appeared to be under polygenic control, including a strong H-2-linked factor. No linkage was found with five different isozyme loci, with theIg-l locus or with C5 serum activity. Also in vivo resistance showed strong linkage with theH-2 complex. In (A x CBA)F₁ x A backcross mice, a weak linkage was found with the coat color locusC. There was a correlation between in vitro killer activity and in vivo resistance in the same backcross mice. In vivo resistance was particularly strong in mice that combined theH-2 ^b -linked resistance factor with a high cytolytic activity in vitro.     

Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A^k antibody to C3H/He (H-2^k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2^d) mice were treated with this antibody. Administration of anti-I-A^k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A^b, I-A^d, I-E^d, and I-E^k molecules resulted in decreased NK activity in B10.D2 (H-2^d) but not in B10.BR (H-2^k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A^k treatment. Removal of adherent cells did not restore NK activity of anti-I-A^k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1⁺ cells from nylon-wool nonadherent SC of mice treated with anti-I-A^k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.     

The effect of the Cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells.

The resistance of mice to lethal infection by murine CMV (MCMV) is under complex host genetic control with contributions from both H-2 and non-H-2 genes. We have previously shown that an autosomal, non-MHC encoded gene, Cmv-1, controls MCMV replication in the spleen. We have investigated the mechanism by which the Cmv-1 resistance gene confers protection against MCMV infection. Using H-2 compatible irradiation bone marrow chimeras, the enhanced resistance to MCMV infection that is associated with the Cmv-1l allele in the C57BL background was shown to be mediated by an irradiation-sensitive bone marrow-derived cell population, or a factor produced by these cells. The lack of correlation between serum IFN titers and the strain distribution pattern of Cmv-1 in CXB recombinant inbred mouse strains suggests that IFN does not mediate resistance conferred by this gene. Similarly, the lack of effect of in vivo depletion of mature CD4+ and CD8+ T cells on virus replication in C57BL/6J mice indicates that T cells are unlikely to be involved. In contrast, in vivo depletion of NK cells by injection of the anti-NK1.1 mAb PK136 abrogated restricted splenic virus replication in C57BL/6J----BALB.B chimeric mice and in the Cmv-1l CXB strains. These data indicate that the effect of the Cmv-1 gene is mediated by NK cells. The significant augmentation in NK cell activity after MCMV infection of the susceptible Cmv-1h strains (BALB/cBy), CXBG/By, CXBH/By, CXBI/By, and CXBK/By) indicates the existence in these mice of NK cells that are functionally and phenotypically distinct from those in Cmv-1l strains. NK cells present in the Cmv-1h strains are unable to restrict efficiently splenic MCMV replication in vivo, possibly due to a lack of specificity for virus-infected target cells. Finally, flow cytometric analysis of NK1-1 expression in CXB and BXD RI mice together with MCMV replication studies in the BXD RI strains indicate that Cmv-1 is closely linked to NK1.1 and other loci that reside on a distal segment of murine chromosome 6 in a region that has recently been defined as the natural killer complex.     

Expression of subregion and haplotype preference for H-2Kk restricted cytotoxic T-cell responses in vivo and at the level of the precursor frequencies

Several strains of mice bearing the H-2K^k allele were found to generate in vivo strong CTL responses against TNP-haptenated syngeneic cells, while several other strains of mice were found to generate comparably weak or no responses. C3H × DBA/2)F1 mice (H-2^k × H-2^d) and A/J mice with the recombinant haplotype $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ generated CTL responses in vivo that were completely restricted toward the H-2^k haplotype or the K end of the $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ haplotype, respectively. The CTL activity of C3H × DBA/2)F1 and A/J mice against haptenated H-2^k targets was found to be more than 25-fold higher than the CTL activity on H-2^d targets. The CTL responses in vitro under macroculture conditions showed, on the other hand, only a 3- to 6-fold higher cytotoxic activity against the haptenated H-2^k targets as compared with haptenated allogeneic or H-2^d targets; and limiting dilution experiments in microcultures revealed that the CTL precursor frequencies were only 2- to 3-fold smaller for TNP-haptenated H-2^d or haptenated allogeneic targets than for haptenated H-2^k target cells. This indicated that sufficient numbers of H-2^d-restricted and allorestricted CTL precursors were actually present in these strains, but did not develop detectable cytotoxic activity in vivo. The exceptional property of the H-2^k haplotype is, therefore, only partly determined by a difference in the CTL precursor frequencies, and to the larger extent determined at the level of the activation of the CTL response.     

Natural resistance against hematopoietic cells in lethally-irradiated mice infected with Friend leukemia virus

Summary The influence of in vivo infection with the polycythemic substrain of Friend leukemia virus on noninducible (natural) resistance against allogeneic normal or malignant grafts was studied in lethally irradiated mice. Parallel studies were performed on the NK system in the same experimental conditions. The results indicate that FLV-P infection of mice with full (DBA/2) vs partial (BALB/c and CD2F1) susceptibility did not suppress their in vivo natural resistance against bone marrow or El-4 leukemia cells. On the other hand, a decline in NK activity paralleled the progression of leukemic disease in the more susceptible DBA/2 hosts. Abbreviations used: FLV-P, N-tropic polycythemic substrain of Friend Leukemia Virus Complex; NR, natural resistance; NR in vivo, natural resistance against normal or malignant hemopoietic grafts occurring in vivo in lethally irradiated mice; NK, natural killer; (¹²⁵I)IUdR, ¹²⁵I-labeled 5-iodio-2-deoxyuridine; IV, intravenous     

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2^b) and P815-X2 (H-2^d), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2^d) and C57BL/6J (H-2^b) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2^k) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cell's ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.     

Natural immunity to grafts of FLD-3 erythroleukemia cells by irradiated mice

Mice were irradiated and infused with BALB/c Friend virus-induced FLD-3 erythroleukemia cells. Growth of the cells was estimated by measuring splenic incorporation of 5-iodo-2'-deoxyuridine-125I 5 days after cell transfer. BALB/cJ and C3H mice were 'poor responders' in that FLD-3 cells grew well in their spleens, while mice of other strains were 'good responders', resisting the growth of FLD-3 cells. No H-2 or Fv genetic locus was associated with resistance. Athymic nude mice and mice depleted of marrow tissue by 89Sr or estradiol resisted FLD-3 cells, indicating that the effectors were thymus- and marrow-independent. Silica, carrageenan and Propionibacterium acnes organisms all altered resistance, suggesting a function of macrophages. Neither interferon nor anti-interferon serum treatment altered resistance. Anti-asialo GM1 serum inhibited resistance to FLD-3 cells in vivo and inhibited natural cytotoxic (NC) activity against FLD-3 cells in vitro. NC (FLD-3) activity was greatly decreased in spleens 3 days after irradiation, in contrast with NK (YAC-1) and NC(WEHI-164.1) activities. Moreover, a 3-day delay in infusion of FLD-3 cells 'synergized' with silica in weakening genetic resistance in vivo. Thus, natural immunity to FLD-3 cells in vivo differs from that of genetic resistance to normal bone marrow cell allografts, and the lysis of FLD-3 cells in vitro seems to be mediated by cells which do not easily fit into the definition of natural killer (NK) or natural cytotoxic (NC) cells.     

Differences in the metastatic potential of two sublines of tumor 3LL selected for resistance to natural NK-like effector cells

Summary Normal syngeneic spleen cells were found to inhibit the local growth of the Lewis lung carcinoma (3LL) when injected together with the tumor cells at a ratio of 100:1 (spleen to tumor cells). The repeated injection of the tumor cells together with spleen cells enventually led to the selection of a tumor cell population whose growth could no longer be inhibited by normal spleen cells. In a previous report from this laboratory, a tumor subpopulation obtained in this manner was shown to display an increased metastatic potential, as well as a decreased sensitivity to natural resistance mechanisms in vivo and NK lysis in vitro. In the present study, we attempted to characterize the spleen cell population which mediated this selection process. We found that spleen cells depleted of T cells, B cells, or adherent macrophages retained their ability to inhibit tumor growth and select a resistant line in vivo. Subsequently, two tumor sublines derived by continuous in vivo passage of the parental tumor line with either unfractionated or nylon woll-non-adherent spleen cells were characterized. It was found that whereas both sublines were resistant to growth inhibition by normal spleen cells, only the subline derived from continuous passage with unfractionated spleen cells showed a reduction in the density of H-2^b molecules expressed on the cell surface and an enhanced metastatic potency. These results suggest that the resistance of a tumor line to natural killer cells may not always result in an increase in its metastatic potential. Abbreviations used in this paper: DBSS, Dulbecco's balanced salt solution; EM, Dulbecco's modification of Eagle's medium; FACS, Fluorescent activated cell sorter; FITC, fluorecein isothiocyanate; FUDR, 5-fluorodeoxyuridine; i.f.p., intrafootpad; Ig, immunoglobulins; IP, intraperitoneal; ¹²⁵IUDR, [¹²⁵I].iodo-2-deoxyuridine; NK, natural killer; N. Tx, neonatally thymectomized; PBS, phosphate-buffered saline; SC, subcutaneously     

Induction of nonspecific killer cells by delayed-type hypersensitivity against soluble protein antigens in murine peritoneal cavities

Summary We induced nonspecific killer cells in the local site of delayed-type hypersensitivity against keyhole limpet hemocyanin or ovalbumin. Delayed-type hypersensitivity was induced in the peritoneal cavities of mice, and peritoneal exudate cells (PEC) were collected. These PEC were found to have killer activity toward SP2 and YAC-1 cells (target cells susceptible to natural killer cells) by 4-h ⁵¹Cr-release assays. The induction of killer activity in PEC was observed in parallel with the eliciting of delayed-type hypersensitivity in the peritoneal cavity, in which the killer activity was maximum 24–48 h after the antigen challenge, but was not induced in nu/nu mice and was induced in an antigen-specific way. These killer cells did not adhere to nylon wool and had Thy1 and asialo-GM1 antigens on their surfaces. Their precursor cells were also asialo-GM1-positive. These findings indicate that the killer cells probably belong to the NK cell lineage. Results of tumor challenge experiments showed that these killer cells had an antitumor effect in vivo as well as in vitro.     

H-2-linked genes determine the level of the primary in vitro anti-Mls response

The level of cell proliferation and interleukin-2 (IL-2) production observed in an anti-Mls mixed lymphocyte reaction between spleen cells from H-2 compatible, Mls incompatible mouse strains is determined by the H-2 haplotype of the mouse combination. Thus, while AKR (H-2 ^k) spleen cells stimulated strong M1s^a responses in H-2^k responder cells, AKR H-2b spleen cells stimulated no or negligible M1s^a responses in responder cells from H-2 ^bmouse strains. This effect was observed at the levels of IL-2 production and cell proliferation. The magnitude of the response observed using F₁ (H-2 ^k/H-2 ^b) responder cells was found to be a function of stimulator rather than responder cells. The poor stimulatory capacity of AKRH-2 ^bspleen cells was also shown not to be due to the loss of the stimulatory Mls ^aallele during the construction of the congenic strain from AKR and C57BL/6 parental strains. Using stimulator cells from a second series of congenic mice, we found H-2 ^b(strain DLLP) again to represent a poorly Mls^a stimulatory H-2 haplotype. In addition, H-2^q (DBA/1) cells displayed very poor Mls^a stimulatory potential while H-2^d (D1.C) cells were efficient Mls^a stimulators. Again the effect was shown to be at the level of the stimulator cells. In toto, our findings indicate that the H-2 ^kand H-2 ^dhaplotypes encode strong Mls^a stimulatory potential while the H-2 ^band H-2 ^qhaplotypes determine poor Mls^a stimulatory potential in primary in vitro responses, measured as cell proliferation and IL-2 production.Abbreviations used in this paper: CTL cytotoxic T lymphocyte - IL-1 interleukin-1 - IL-2 interleukin-2 - MLR mixed lymphocyte reaction - NMS normal mouse serum     

Natural resistance of irradiated 129-strain mice to bone marrow allografts: Genetic control by theH-2K region

A major genetic determinant of natural resistance to bone marrow allografts, designated asHh-3, was mapped to theH-2K region. This gene may code for or regulate the expression of cell surface structures selectively expressed on donor hemopoietic cells and recognized by naturally occurring cytotoxic effectors. Resistance was observed as failure of donor cell growth in the spleen of irradiated 129-strain (H-2 ^bc ) recipients of H-2^k bone marrow cells. The mapping was accomplished by substituting donor cells bearingk alleles throughout theH-2 complex with cells of recombinant mouse lines bearingk alleles at definedH-2 regions. The host antigraft reaction underlying resistance was abrogated by pretreating 129-strain mice with either rabbit antimouse lymphocyte serum or the antimacrophage agent silica. Grafting of H-2K^k cells into mice ancestrally unrelated to 129 but sharing theH-2 ^bc or the similarH-2 ^b haplotype, and intoH-2 ^b/k ,H-2 ^k/bc , andH-2 ^k/d F₁ hybrids revealed that resistance was unique to 129 mice, since mice of the other strains, including F₁ hybrids, were susceptible to the grafts. Thus,Hh-3 incompatibility was a necessary but insufficient condition for the manifestation of allogeneic resistance; other genetic factors not associated withH-2 conferred responder status to 129-strain mice and nonresponder status to D1.LP, B10.129(6M), B10, B6, and possibly to F₁ hybrid mice. The possible relationships between allogeneic resistance to H-2^k marrow grafts, hybrid resistance to H-2^k lymphomas, and F₁ hybrid antiparental H-2^k cytotoxicity induced in vitro are discussed.     

The regulation of immune responses to multiple non-H-2 antigens on tumor cells: I. Differential responses of BALB/c F1 hybrids to the P815 mastocytoma in vivo

Factors influencing host resistance to the growth of a tumor bearing many mismatched minor histocompatibility antigens (MiHA) were studied. BALB/c (H-2^d) and several of its F₁ hybrids were injected intraperitoneally with DBA/2 (H-2^d) P815 tumor cells. Compared to BALB/c, which was moderately susceptible, F₁ hybrids of BALB/c with CBA, AKR, C3H.OH, and BIO H-2-congenic strains were highly susceptible, whereas hybrids of BALB/ c with A, A.SW, and BALB.B strains were quite resistant. Susceptibility was observed only with the intraperitoneally injected tumor, since both BALB/c and (CBA x BALB/c)F₁ were resistant to the same tumor injected subcutaneously, and survival times of DBA/2 skin grafts did not differ between susceptible and resistant strains. Susceptibility was in part a function of the number of MiHA incompatibilities between tumor and host although the specific loci involved could not be identified. For example, susceptible (CBA x BALB/c)F₁ hybrids probably shared certain MiHA with DBA/2 which BALB/c lacked, and which therefore subtracted from the net antigenic strength of the tumor in the hybrid, compared to its strength in BALB/ c. This interpretation was supported by in vitro studies which confirmed that the susceptible hybrids shared more MiHA with DBA/2, than did the resistant hybrids. Resistance was at least partially regulated by the host H-2 genotype, as shown by the observation that (BALB/ c x BALB.B)F₁ (H-2^d/b) mice were significantly more resistant than BALB/c. Segregation studies of the resistant (BALB/c x A)F₁ hybrids, indicated that in addition to H-2, a nonH-2 gene in the A background was operating to confer resistance. Thus the factors influencing susceptibility to the MiHA-incompatible tumor were: (i) site of injection; (ii) the combined strength of the disparate MiHA; (iii) the host H-2 genotype; and (iv) at least one host nonH-2 gene conferring increased responsiveness.     

Evidence for differentiation of NK1+ cells into cytotoxic T cells during acute rejection of allogeneic bone marrow grafts

The ability of lethally irradiated C57BL/6 mice to acutely reject H-2^d bone marrow is due to a lymphocyte population that is NK1⁺, ASGM1⁺, CD4^–, CD8^–, CD3⁺. Transfer of spleen cells from C57BL/6 mice expressing these antigens into nonresponder 129 mice adoptively transfers the ability to reject H-2^d marrow grafts. The specificity of this rejection maps to the H-2D major histocompatibility complex (MHC) region. Transplantation of high doses of H-2^d marrow into C57BL/6 overrides the acute rejection mechanism leading to graft survival. During growth of the graft, a cytolytic activity develops that is due to ASGM1⁺, CD8⁺ cytolytic T lymphocytes (CTLs) with H-2L^d specificity. The possibility that the ASGM1⁺, CD8⁺ CTLs are descendents of the CD3⁺, NK1⁺, ASGM1⁺, CD8^– cells responsible for acute rejection is investigated by adoptive cell transfer experiments. We show that beige mice that lack NK1⁺ cells as well as the ability to acutely reject H-2^d marrow fail to generate specific CTLs after transplantation with a high dose of H-2^d marrow. Transfer of highly purified NK1⁺ cells from B6.PL-Ly-2 ^a /Ly-3 ^a (Lyt-2.1) into beige mice together with H-2^d marrow leads to generation of Lyt-2.1 CTLs from donor NK1⁺ cells. These results show that specific CTLs are generated from NK1⁺ cells during acute marrow graft rejection. Offprint requests to: G. Dennert.     

Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy

Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T. DeKretser and B. Livett, Nature (London), 263, 682, 1976). Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M. Hansson, K. Karre, R. Kiessling, J. Roder, B. Anderson, and P. Hayry, J. Immunol., 123, 765, 1979). The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied. Spleen cells from normal (+/+) and dystrophic ($\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$) male C57BL/6J mice 8–10 weeks old were passed over nylon wool and the nonadherent cells were incubated with ⁵¹Cr-labeled YAC-1 lymphoma target cells or thymocytes in a ⁵¹Cr-release assay. Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice. Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells. Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets. Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets. Target cellbinding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets. The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group. Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group. Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell. Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages. Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity.     

Immunogenetic basis for immunologic privilege in the anterior chamber of the eye

DBA/2 mastocytoma (P815) cells are able to grow when inoculated into the anterior chamber of eyes of histoincompatible mice. Tumor growth is unrestrained in recipient mice differing from the DBA/2 strain at multiple minor H loci, but sharing the H-2 ^d haplotype; progressive tumor growth in these animals involves the entire eye, invades the orbit and kills the hosts by extension into the cranial vault. Alternatively, P815 cells grow initially but are unable to sustain continued growth in the anterior chambers of recipient mice differing from DBA/2 at the H-2 complex. Recipients differing from DBA/2 at either the K or D regions of H-2 develop anti-DBA/2 immunity that destroys the intraocular tumor within 20 days of inoculation. Severe inflammatory reactions in these eyes produce innocent bystander destruction of ocular tissue and produce blindness. Recipients differing from DBA/2 at both K and D regions of H-2 also mount vigorous anti-DBA/2 immunity that destroys the intraocular tumor. In these instances, the nonspecific component of the rejection reaction is minimal: when the tumor cells are destroyed, the eyes are restored to anatomic and functional integrity. These results indicate that immunologic privilege in the anterior chamber of the eye is afforded to allogeneic tissues to varing degrees depending upon allodisparity at H-2 loci encoding class I MHC products. The results further imply that the precision of the alloimmune response may be under the control of these same MHC products.     

Pronounced antitumor effect of LAK-like cells induced in the peritoneal cavity of mice after intraperitoneal injection of OK-432, a killed Streptococcal preparation

Summary More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i. p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i. p. injection of the admixture of the nonadherent PEC (1×10⁷ cells) with BAMC-1 cells (1×10⁵) survived for more than 60 days. When the same nonadherent PEC (1×10⁷ cells) were i. p. transferred adoptively 1 day after the inoculation of 1×10⁵ BAMC-1 tumor cells, again all mice survived.When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations.These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.     

Experimental allergic orchitis in mice

Inbred strains of mice were studied for their susceptibility to the induction of experimental allergic orchitis after sensitization with mouse testicular homogenate in complete Freund's adjuvant accompanied by injections of extract from Bordetella pertussis. Susceptibility to autoimmune orchitis was found to be linked to the major histocompatibility complex in BALB/c and C57BL/10 mice and mapped to genes encoded within the H-2D ^dregion. In five of six groups of bidirectional (susceptible × resistant) F₁ hybrids, H-2D ^d-linked susceptibility was inherited as a dominant autosomal trait. However, in (BALB/cByJ × DBA/2J)F₁ and (DBA/2J × BALB/cByJ)F₁ hybrids, dominant autosomal resistance to the induction of autoimmune orchitis was observed. Backcross analysis between the resistant F₁ hybrid and the susceptible BALB/cByJ parent suggests that a single independently segregating DBA/2J locus is capable of negating H-2D ^d-linked susceptibility, and controls resistance to the induction of autoimmune orchitis.Abbreviations used in this paper BP extract Bordetella pertussis extract - CFA complete Freund's adjuvant - EAO experimental allergic orchitis - Ir immune response - MHC major histocompatibility complex - MLH mouse liver homogenate - MTH mouse testis homogenate - PI pathology index     

Immunological and genetic requirements for the parental hemopoietic takeover reaction in adult,H-2-incompatible parent-F1 hybrid parabiosis

Parabiosis of adult DBA/2J (H-2 ^d ) mice with adult (DBA/2J× CSH/HeJ)F₁ (H-2 ^d /H-2 ^k ) mice results in survival beyond 100 days in 44% of such pairs, induction ofin situ unresponsiveness to C3H/HeJ skin, and the complete takeover of the erythroid system of the F₁ by parental cells. However, in vitro responsiveness to C3H/HeJ cells remains. Dye exclusion cytotoxicity assays establish the absence ofF ₁ lymphoid cells in the spleens and bone marrow of both partners. The parental takeover of the erythroid system of the F₁ partner requires immune recognition of the hybrid's alloantigens, because this takeover is not seen with tolerant parental cells. PartialH-2 differences (on the C3H background) influence both survival and the takeover reaction when incorporated into parabioses with DBA/2J partners. When only theK andI subregions ofH-2 were targets of the parental response, 58% of parabionts survived, with complete parental hemopoietic takeover. When onlyH-2D was the target, 83% of parabionts survived, with incomplete hemopoietic takeover. Changing the non-H-2 background of the F₁ target did not significantly affect survival or takeover, while substituting a differentH-2 ^d parental strain (BALB/c) eliminated survival altogether.Thus the parabiont takeover reaction encompasses the lymphoid and hemopoietic systems, requires immunorecognition of the target alloantigens, and seems to require a strongH-2 difference for its induction.     

Genetic control of murine hemagglutinin formation to H-2.5

When B10.D2 (H-2^d) mice are immunized with lymphoid cells from C57B1/10 (H-2 ^d ) and their antisera tested against B10.A (H-2 ^a ) target cells, only antibodies to H-2.5 are measured. The same is true for immunization of DBA/2 (H-2 ^d ) mice when their antisera are absorbed with B10.D2 cells prior to testing. Irrespective of the dose of immunogen administered, the primary hemagglutinin response of B10.D2 mice is significantly lower than that of DBA/2 mice and (B10.D2 × DBA/2)F₁ hybrids, but the secondary responses are similar. The low responsiveness of B10.D2 mice appears to be determined by a single dominant gene with incomplete penetrance; the gene is not linked to eitherH- 2, Hc, or the immunoglobulin allotype loci. In addition, the H-2.5 hemagglutinin response is susceptible to nongenetic influences. When antisera from B10.D2, devoid of H-2.5 hemagglutinins, were assayed in a complement-mediated cytotoxic test, they contained almost as much anti-H-2.5 activity as did the antisera from DBA/2 mice or (B10.D2 × DBA/2)F₁ hybrids. The possibility is discussed that the locus responsible for the deficient primary hemagglutinin response of B 10.D2 may not be determinant-specific but may affect hemagglutinin responses in general.     

Genetic control of contact sensitivity in mice: Effect ofH-2 and nonH-2 loci

The genetic control of delayed-type hypersensitivity in mice was investigated by contact sensitization with picryl chloride. Distribution patterns of contact sensitivity in 11 inbred strains of mice showed significant differences among strains. Comparison of levels of response between congenic-resistant lines and their inbred partners, at 9 to 11 weeks of age, revealed a clear association betweenH-2 haplotype and the magnitude of response. Testing ofH-2 recombinants further suggested the influence of two genes mapping at either end of theH-2 complex. While theH-2K ^d andH-2D ^k alleles were associated with a high response, theH-2K ^k ,H-2K ^b ,H-2D ^d , andH-2D ^b alleles were associated with a low response. Analysis of the ontogeny of response suggested that theH-2 haplotype manifests its effect through the maturation of contact sensitivity. On both the C57BL/6By and C57BL/10Sn backgrounds, theH-2 ^d haplotype was associated with early maturation of response, while theH-2 ^b haplotype was associated with late maturation. Analysis of the response of congenic lines with different genetic backgrounds and of CXB recombinant-inbred lines further revealed the marked effects of yet other genes on this trait.