Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

Adoptive immunotherapy of a BALB/c lymphoma by syngeneic anti-DBA/2 immune lymphoid cells: Characterization of the effector population and evidence for the role of the host's non-T cells

Summary It has been previously shown that the BALB/c lymphoma YC8 is susceptible to lysis by syngeneic anti-DBA/2 lymphocytes and that YC8-bearing BALB/c mice can be cured by adoptive transfer of such immune effectors. In this study in vivo and in vitro functions of the curative immune lymphocytes have been evaluated together with the role of the host immune system in the mechanism of tumor eradication. It was found that the curative anti-DBA/2 lymphocytes were not directly cytotoxic to YC8 cells although they developed into YC8-lytic cells after in vitro restimulation with YC8. In vivo, the immune lymphocytes were able to mediate a tumor-specific delayed type hypersensitivity reaction against YC8 but had a low tumor-neutralizing activity in the Winn assay. Proliferation of infused BALB/c anti-DBA/2 lymphocytes was necessary for the in vivo therapeutic effect, since irradiation of effector cells or treatment of the donor immune lymphocytes with vinblastine abolished their curative capacity. Immunodepression of the T cell compartment of the prospective tumor-bearing animals by thymectomy plus irradiation or its abrogation in B mice (thymectomized, lethally irradiated, and reconstituted with fetal liver cells) did not interfere with the therapeutic effect of the transferred anti-DBA/2 lymphocytes. Blocking the macrophage functions of the host by carrageenan, however, abolished the therapeutic effect of immune lymphocytes. These data indicate that a radiation-resistant, non-T cell is involved in the tumor eradication induced by anti-DBA/2 lymphocytes. It was also shown that cured mice, tested 90 days after therapy, become resistant to 5×10³ LD₈₀ YC8 cells and that this resistance was due to the presence of memory cells derived from the transferred and not from the host lymphocyte population.     

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of ¹²⁵I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.     

Differential resistance to growth of a tumor expressing incompatible minor alloantigens reflects regulatory influences rather than differences in anti-minor-CTL-P frequencies

In previous studies it was found that BALB/c (H-2d) was more susceptible than (BALB/c X A)F1 (H-2d X H-2a) to a tumor bearing multiple mismatched minor histocompatibility antigens, the DBA/2 (H-2d) mastocytoma P815, and that this resistance was H-2 linked. In the present studies the immunologic basis of this effect was examined by comparing the cytotoxic-T-lymphocyte (CTL) responses of BALB/c with those of (BALB/c X A)F1. Despite the BALB/c X A)F1's 34-fold greater resistance to P815 in vivo, the numbers of effector cell precursors were found to be similar in the two hosts as shown by (a) similar anti-P815 CTL responses in vitro with T-cell growth factor, (b) similar secondary anti-DBA/2 MiHA responses after in vivo priming with irradiated P815, and (c) similar frequencies of anti-DBA/2 CTL precursors by limiting-dilution analysis. However, priming with proliferating P815 in vivo revealed a defect in the BALB/c animals: Spleen cells from such animals were unable to control the growth of contaminating P815 cells in vitro or to mount strong secondary CTL responses to DBA/2 antigens. The defective priming of BALB/c could be corrected when DBA/2 spleen cells were added to the P815 inoculum. This impaired priming by living tumor cells was not seen in (BALB/c X A)F1. It is concluded that the use of living P815 tumor cells revealed a defect in immunoregulation in BALB/c mice, which rendered them susceptible to tumor growth in spite of apparently adequate numbers of anti-minor-CTL precursors. How the additional H-2 products expressed in the (BALB/c X A)F1 might correct this defect is discussed.     

Immunodominance in the immune response to “multiple” histocompatibility antigens

Cytotoxic effector T cells putatively specific for multiple non-H-2 histocompatibility (H) antigens were generated by immunizing and boosting C57BL/6 and B6.C-H-2 ^dmice with BALB.B and BALB/c stimulator cells, respectively. The generated effectors were tested for cell-mediated lympholysis on a panel of targets whose BALB/c-derived non-H-2 H antigens were donated by CXB recombinant inbred mice. The spectrum of reactivity of cytotoxic effector T cells with CXB targets demonstrated that the effectors did not recognize multiple H antigens but rather preferentially recognized a single immunodominant non-H-2 H antigen. The identity of the immunodominant H antigen was determined by the H-2 genotype of the stimulator cells when (B6 × B6.C-H-2 ^d)F ₁ cytotoxic effectors were tested. These observations indicate that despite the fact that responders were challenged with more than 40 individual non-H-2 H antigens, they preferentially responded to a single immunodominant antigen.     

Differential responsiveness to MIs locus antigens in graft-versus-host and host-versus-graft reactions

After (semi)allogeneic transplantation of lymphoid cells into lethally irradiated mice, the development of anti-host directed T effector cells can be demonstrated by means of a simple delayed-type hypersensitivity (DTH) assay. Using this assay we have shown that in H-2 compatible combinations Mls locus antigens can induce the generation of such T effector cells during a graft-versus-host (GvH) reaction. Other non-H-2 alloantigens are probably of minor importance. The capacity of Mls locus antigens to induce distinct anti-host DTH reactivity correlated with the capacity to induce a one-way mixed lymphocyte culture (MLC) response. Mls^a and Mls^c locus antigens initiated a positive MLC response as well as distinct GvH-related DTH reactivity. On the other hand, in the combination DBA/2 versus (BALB/c × DBA/2) F₁, the Mls^b locus antigen was not able to initiate in vitro proliferation, a lack of response which coincided with a marginal and short-lasting GvH-related DTH reactivity. In contrast, the host-versus-graft (HvG) DTH reaction of BALB/c and DBA/2 mice to subcutaneously injected (BALB/c × DBA/2) F₁ spleen cells was equally strong. Here antigens other than those coded for by the Mls locus were mainly responsible for the antigraft DTH response. These results suggest that T effector cells generated in GvH and HvG reactions are specific for largely different sets of minor histocompatibility antigens, with a selective stimulation by Mls locus antigens under GvH conditions.     

Intratumoral low-dose interleukin-2 induces rejection of distant solid tumour

Summary This study shows that local tumour treatment with low-dose recombinant interleukin-2 (IL-2) can mediate rejection of a large distant solid tumour. When SL2 lymphoma cells were injected intraperitoneally (i.p.) in syngeneic DBA/2 mice on day 0, 70% of these mice were cured by daily i. p. injections with 20 000 units IL-2 on days 10–14. After injecting mice with SL2 both i.p. and subcutaneously (s. c.) on the flank, 50% of the mice treated i.p. with low-dose IL-2 rejected both the i.p. tumour and the large distant s.c. tumour. In contrast, i.p. IL-2 treatment on days 10–14 cured fewer than 10% of the mice bearing only a s. c. SL2 tumour. The described IL-2 immunotherapy also caused systemic tumour rejection in mice bearing both ascitic and solid P815 mastocytoma. Thus it was shown that low-dose IL-2 can induce systemic tumour rejection, when injected at a site of tumour growth. Interleukin-2-induced rejection of s. c. SL2 tumour was highly specific, as mice that were rejecting i.p. and solid s. c. SL2 lymphoma did not reject solid P815 mastocytoma, which was injected s.c. simultaneously on the other flank. Furthermore, solid s.c. tumours consisting of mixtures of SL2 and P815 were not rejected in mice that rejected i.p. SL2 or P815. We conclude that intratumoral injections of low-dose IL-2 can enhance an ongoing weak immune reaction against the tumour resulting in systemic tumour rejection.     

Immunogenetic studies of spontaneous abortion in mice. Preimmunization of females with allogeneic cells

CBA females (H-2k) mated with DBA2 males (H-2d) exhibit a high rate of fetal resorption (30%) when compared with the CBA female BALB/c male, CBA female/CBA male, DBA2 female/CBA male, DBA2 female/DBA2 male combinations (6 to 8%). Preimmunization of CBA females with spleen cells from DBA2, BALB/c, or CBA males were performed in order to test their effects on CBA maternal tolerance of (CBA X DBA2)F1 fetuses. Only preimmunization with BALB/c male cells was effective in decreasing resorption; cells from BALB/c females had no effect. In order to further test 1) the role of non-MHC-encoded antigens present in the BALB/c male background, 2) the necessity of an additional H-2 difference, and 3) whether or not the phenomenon is H-2d restricted, preimmunizations were performed by using cells from congenic BALB/k (H-2k), BALB/b (H-2b), or BALB/c (H-2d). Only the latter treatment was efficient, which suggests that the paternal H-2d haplotype must be presented in synergy with some non-MHC-encoded antigens in the BALB/c male background. Immunogenetic studies with cells from nine recombinant inbred strains that reassorted DBA2 and BALB/c genomes showed that three of them behave like BALB/c and six like DBA2. This would suggest that the genetic determinism of this phenomenon is simple.     

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1–3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20 000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 × 10⁴ cells injected i.p.) consisted of i.p. injections of 5000 or 20 000 U IL-2/mouse given on days 10–14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity. Received: 16 July 1998 / Accepted: 5 October 1998     

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.     

T lymphocyte responses to non-H-2 histocompatibility antigens. I. Role of Ly-1+2+ T cells as cytotoxic effectors and requirement for Ly-1+2+ T cells for optimal generation of cytotoxic effectors

Cytotoxic effector T cells specific for non-H-2 histocompatibility (H) antigens were examined for phenotypic expression of lymphocyte differentiation (Ly) antigens. Virtually all H-Y-specific cytotoxic effectors generated in mixed lymphocyte culture were Ly-1+2+ T cells. H-3-specific effectors comprised both Ly-1+2+ and Ly-1-2+ T cells. However, cytotoxic effectors specific for multiple non-H-2 H antigens were predominantly Ly-1-2+ T cells. The optimal generation of H-Y- and H-3-specific effectors required Ly-1+2+ T cells; optimal generation of multiple non-H-2 H antigen-specific effectors required an interaction between Ly-1+2- and Ly-1-2+ T cells. These observations suggest that the identity of the target H antigen in part determines the Ly type of responsive T cells. Our observations suggest that 2 alternative pathways of T cell response exist for non-H-2 H antigens. The first pathway involves an interaction between Ly-1+2- helper T cells and Ly-1-2+ cytotoxic effector precursors. The 2nd pathway simply involves the response of Ly-1+2+ T cells proliferating and generating H antigen-specific cytotoxic effectors.     

Sex-dependent effects of non-H-2 loci on lethal GVH reaction induced across an H-2 barrier

We have studied the influence of DBA/2 non-H-2 antigens on the lethal graft-versus-host reaction (GVHR) developed across an H-2 barrier. (DBA/2 x B10.D2)F₁ x B10.D2 (H-2 ^d) backcross (BC) mice were typed for their allelic constitution at nine genetically independent chromosome markers and used as individual cell donors simultaneously for two to three (DBA/2 X B10.D2)F₁ recipients incompatible for DBA/2 non-H-2 antigens alone and two to three (DBA/2 x B10.BR)F₁ recipients incompatible for DBA/2 non-H-2 antigens and H-2^k. The results showed that, when compared with that developed in a control group incompatible for H-2 ^kalone [B10.D2(B10.D2xB10.BR)F₁], the GVHR mortality seen in the presence of an additional incompatibility for DBA/2 non-H-2 antigens [(DBA/2 X B10.BR)F₁recipients] is significantly delayed but only in female mice. An analysis of individual BC donors indicated that this protective effect of DBA/2 non-H-2 antigens correlates with incompatibility for gene(s) linked to the Pgm-1 chromosome marker. In contrast, incompatibility for gene(s) linked to Mod-1 and Es-3 markers accelerates GVHR mortality, but only in male mice. Finally, the results obtained with (DBA/2 x B10.D2)F₁ and (DBA/2 x B10.BR)F₁ recipients were compared; they showed that the intensity of the GVHR developed by cells from individual BC donors against a given set of DBA/2 non-H-2 antigens correlates well with that developed by the same BC donor against the same set of non-H-2 antigens plus H-2^k. We conclude that certain non-H-2 genes (and antigens) can modulate the intensity of the GVHR developed across an H-2 barrier. The number of such genes is probably great; their effects are strong and complex, and can be sex-dependent.     

Genetic control of cross-reactive cytotoxic T-lymphocyte responses to a BALB/c tumor

Using a segregation analysis we have determined that the cross-reactive response to the DBA/2 tumor P815 by CTL from BALB/c mice immunized with a BALB/c plasmacytoma (MOPC-167) is controlled by a single gene. The gene responsible is closely linked to the dilute coat color locus on chromosome 9. In contrast, the cross-reactive response to the DBA/2 tumor L5178Y by DBA/2 anti-MOPC-167 CTL appears to be controlled by two or more genes.Abbreviations used in this paper BXD RI C57BL/6 × DBA/2 recombinant inbred - CD2F1 (BALB/c × DBA/2)F₁ - CTL cytotoxic thymus-derived lymphocyte - IUdR 5 iododeoxyuridine - PEC peritoneal exudate cell     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

A fluorescence study on the mobility of surface antigens of untreated tumor cells and of tumor cells undergoing cell-mediated lysis

Heterologous (rabbit) antibodies were raised against murine P-815 mastocytoma cells of DBA/2 origin. Antisera and IgG preparations were highly cytotoxic, whereas Fab fragments thereof lost all activity. Fab fragments also showed a much lower avidity than IgG, both for tumor and normal DBA/2 and C57 spleen cells as measured by the release of iodinated Fab and IgG. Both preparations bound specifically to P-815 cells since they were capable of inhibiting T cell-mediated target cell lysis. The binding of IgG and monovalent Fab fragments was studied by fluorescence. Rhodamine-coupled IgG bound homogeneously in the cold and quickly formed patches upon warming but did not form caps even after prolonged incubation at 37 degrees C. Rhodamine-coupled Fab fragments also bound homogeneously. Their distribution was unaltered after incubation at 37 degrees C even when tumor cells formed uropod-like tails. Fab fragments, however, could be induced to cap with a second and third antibody layer. P-815 cells labeled with rhodamine-coupled Fab fragments were incubated with cytolytic T cells (CTL). The conjugates formed between CTL and fluorescent target cells were observed. No gross redistribution of surface antigens on target cells was observed even at late stages of the lytic process. CTL, therefore, do not seem to operate via a redistribution of surface antigens.     

A stable single-chain variable fragment expressing transfectoma demonstrates induction of idiotype-specific cytotoxic T-cells during early growth stages of a murine B-lymphoma

The idiotypic determinants associated with the variable regions of antibody molecules are known to function as tumor-associated antigens (TAAs). However, there is no clear-cut evidence documenting their efficacy in inducing TAA-specific cytotoxic T-lymphocytes (CTLs). In most previous studies, idiopeptides were implicated in elicitation of TAA-specific CD4+ T-cells. Using a murine B-cell lymphoma, 2C3, we earlier demonstrated induction of splenic CD4+ and CD8+ T-lymphocytes directed to idiotypic Ig of the tumor. In the present study, we provide more direct evidence of the existence of Id-specific CTLs in the spleens of 2C3 bearing BALB/c mice using an scFv-transfectoma, P815A4, as a target. While both P815A4 and 2C3 cells were equally susceptible to cytolysis by the effector cells, lysis was evident only during early tumor progression. Moribund animals at the late stage of tumor growth failed to demonstrate any significant cytotoxic immune response against either tumor. Antibodies to MHC class I alleles Kd, Dd, Ld, beta2m and CD8 molecules all inhibited cytotoxicity. The CTL population from early tumor-bearers recognized 2C3 tumor in the context of all major H-2d alleles; however, in case of P815A4 cells, it was restricted to Kd and Dd alleles only. Based on these antibody inhibition studies, it appears that the idiopeptides generated in both tumors are in some way different, yet they were recognized equally by CTLs not only from the tumor-bearers but also by CTLs from 2C3-hyperimmune mice. It appears that scFv-containing transfectomas expressing antibody variable region epitopes would be useful for both elucidating CTL-defined idiopeptides and monitoring TAA-specific CTL response in tumor-bearing animals.     

The synergistic action of lipopolysaccharide and muramyl dipeptide in the immunotherapy of DBA/2 mice with mastocytoma P815

The intratumoral administration of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) in combination, but not separately, resulted in necrosis and rejection of subcutaneous P815 mastocytoma nodules in DBA/2 mice with 30 to 40% survival. Previous sensibilization of animals by LPS + MDP, treatment by indomethacin, cyclophosphamide or syngeneic lymphocytes did not augment the immunotherapeutic action of LPS + MDP combination. Reinoculation of P815 cells into cured DBA/2 mice 8 months after the disappearance of the primary tumor led to rejection of new nodules with 50% survival rate. In LPS + MDP immunotherapy of these tumors two stages may be distinguished by a thrombo-necrotic stage and that of development of immunity.     

Analysis of cytotoxic mechanisms induced by minor histocompatibility antigens in the mouse.

Following immunization of BALB/c (H-2^d) mice against the P815Y (H-2^d) mastocytoma, two populations of effector cells could be identified in the spleen, namely, the cytolytic T cell and a cytostatic effector, which was resistant to anti-T-cell serum and complement and appeared to be adherent. Quantitative comparison of the activities of both effectors has been made with the levels of activity obtained following immunization across the major H-2 barrier in C57BL10 (H-2^b) mice. While T-cell activity was significantly lower in BALB/c mice, the non-T-cytostatic activity was greater compared with C57BL mice. Therefore, H-2 antigens do not appear to be essential for the efficient induction of the cytostatic effector.     

Evidence for a B lymphocyte defect underlying the anti-X anti-erythrocyte autoantibody response of NZB mice.

The autoimmune hemolytic anemia of NZB mice is pathogenetically mediated by a genetically prescribed anti-erythrocyte autoantibody response directed to the X erythrocyte autoantigen. The cellular locus of the immunoregulatory defect underlying the anti-X response was explored by adoptively transferring bone marrow cells (BMC) from NZB mice to lethally irradiated histocompatible recipients. Before adoptive transfer, BMC from donor mice were assayed for antigen-binding lymphocytes with receptors for the X autoantigen (X-ABL) by immunocytoadherence assays and for anti-X autoantibody-secreting cells (X-PFC) by plaque-forming cell assays. Twelve weeks after adoptive transfer, splenic lymphocytes from recipient mice were assayed for X-PFC and humoral anti-X autoantibody by Coombs' tests. Transfer of 15 to 30 x 10(6) BMC containing 6 to 12 x 10(3) X-ABL but no X-PFC from 6- to 8-week-old NZB mice to lethally irradiated BALB/c, B10.D2, C57BL/Ks, and DBA/2 mice produced X-PFC in 70% of the recipients. Development of X-PFC was not simply dependent upon available X-ABL since transfer of 15-30 x 10(6) BMC, containing comparable numbers of X-ABL, from BALB/c, B10.D2, C57BL/Ks, or DBA/2 mice to NZB or syngeneic recipients did not produce X-PFC. Transfer of BMC from NZB mice to BALB/c, B10.D2, and DBA/2 mice with weekly administrations of AKR anti-theta antiserum had no effect on the development of X-PFC; Tlymphocyte ablation was evidenced by the absence of theta+ spleen cells. These results suggest that the pathogenetic anti-X response is not genetically prescribed at the level of macrophages, humoral factors, or T cells, but rather appears to be a phenotypic expression of a primary B lymphocyte defect permitting or promoting differentiation of NZB X-ABL.     

Anti-thymocyte autoimmunity in BALB/C mice accompanying immunization to a syngeneic lymphoma.

As immunization of BALB/c mice to the syngeneic P1798 lymphoma is effected by administration of iodoacetamide-modified P1798 cells, serum antibodies appear which are reactive with P1798 and normal BALB/c thymocytes, splenocytes, and peripheral blood lymphocytes. Anti-P1798 serum also cross-reacted with thymocytes from AKR, DBA/2, and C3H mice as well as the allogeneic lymphoma 6C3HED. Anti-P1798 serum was unreactive with the Thy-1 deficient L1210 lymphoma. Multiple absorptions of anti-P1798 serum with normal BALB/c thymocytes or brain or P1798 removed antibodies to P1798 and thymocytes commensurately. Normal BALB/c liver and kidney did not absorb antibody activity. Treatment of a BALB/c splenocyte suspension with anti-Thy 1.2 serum and complement removed the population of spleen cells which were capable of reaction with anti-P1798 serum. The data suggest that antibodies to P1798 and thymocytes are the same and that specificity may be directed toward a Thy-1 related structure but without distinguishing Thy-1.1 and Thy-1.2.