GENETIC CONTROL OF BONE MARROW GRAFT REJECTION : I. DETERMINANT-SPECIFIC DIFFERENCE OF REACTIVITY IN TWO PAIRS OF INBRED MOUSE STRAINS

Transplantation of 5 x 10⁵ DBA/2 (H-2^d) bone marrow cells into irradiated B10 and 129-strain mice (both H-2^b) resulted in graft failure in the first recipient strain and in graft take in the second. Transplantation of B10 (H-2^b) cells into irradiated B10.BR and C3H mice (both H-2^k) also resulted in failure in the congenic B10.BR recipients and take in the C3H mice. Resistance and susceptibility of B10 and 129-strain animals were specific for given H-2 alleles of donor cells. Transplantation of DBA/2 marrow into (B10 x 129)F₂ mice and of B10 marrow into (B10.BR x C3H)F₁ x C3H backcross mice revealed definite genetic control of the graft-rejection process, presumably at the level of alloantigen recognition. Resistance to allografts, or responder status, was conferred upon segregating mice by dominant alleles of two major independent autosomal loci. The effects of the loci were additive. Conversely, susceptibility to allografts, or nonresponder status, was due to the apparently recessive alleles of both loci. None of the genes was closely linked with the markers tf (tufted) and T (brachyury) of linkage group IX, A^w (white-bellied agouti) of linkage group V, Sl (steel) of linkage group IV, and c^ch (chinchilla) and p (pink eye, dilute) of linkage group I. There were suggestions, however, that the regulator genes of marrow graft rejection are either non-H-2 histocompatibility genes or other genetic factors closely linked with them.     

CELLULAR BASIS OF THE GENETIC CONTROL OF IMMUNE RESPONSES TO SYNTHETIC POLYPEPTIDES : II. FREQUENCY OF IMMUNOCOMPETENT PRECURSORS SPECIFIC FOR TWO DISTINCT REGIONS WITHIN (PHE, G)-PRO--L, A SYNTHETIC POLYPEPTIDE DERIVED FROM MULTICHAIN POLYPROLINE, IN INBRED MOUSE STRAINS

DBA/1 mice are high responders to the (Phe, G) determinant of the synthetic polypeptide (Phe, G)-Pro--L, whereas SJL mice respond well to the Pro--L region of this macromolecule (6). In order to determine whether the phenomenon described above is related to the number of antigen-sensitive units detected for both specificities, and whether responses to these determinants can be transferred independently, graded and limiting inocula of spleen cells from SJL, DBA/1, and F₁ donors were injected into X-irradiated, syngeneic, recipient mice with (Phe, G)-Pro--L. By this approach, one antigen-sensitive unit specific for (Phe, G) was detected in 1.7 x 10⁶ and 8.5 x 10⁶ spleen cells from immunized and nonimmunized DBA/1 donors, respectively. In contrast, one (Phe, G) relevant precursor was detected in 20 x 10⁶ SJL spleen cells, irrespective of whether the donors had been immunized. On the other hand, for the Pro--L specificity, one limiting splenic precursor was found in 1.3 x 10⁶ and in 3.4 x 10⁶ cells for immunized and nonimmunized SJL donors, respectively; whereas one response unit was estimated for this determinant in 9.4 x 10⁶ and in 38 x 10⁶ spleen cells from immunized and nonimmunized DBA/1 mice. The findings reported here indicate that the phenotypic expression of the genetic control(s) for immune responsiveness to different immunopotent regions of (Phe, G)-Pro--L is directly correlated with the number of immunocompetent response units detected in two inbred mouse strains. In the spleens of immunized F₁ donors, similar frequencies of one limiting precursor in 3.0 x 10⁶ and in 2.8 x 10⁶ cells were detected for (Phe, G) and Pro--L, respectively. The results of a chi-square test for independence of (Phe, G) and Pro--L responses in F₁ animals is compatible with the hypothesis that the transferred spleen cells limiting the response to (Phe, G)-Pro--L are restricted to generate antibodies specific for only one of the two determinants of this macromolecule.     

CELLULAR BASIS OF THE GENETIC CONTROL OF IMMUNE RESPONSES TO SYNTHETIC POLYPEPTIDES : I. DIFFERENCES IN FREQUENCY OF SPLENIC PRECURSOR CELLS SPECIFIC FOR A SYNTHETIC POLYPEPTIDE DERIVED FROM MULTICHAIN POLYPROLINE ([T, G]-PRO--L) IN HIGH AND LOW RESPONDER INBRED MOUSE STRAINS

SJL mice are high responders to the synthetic multichain polypeptide antigen (T,G)-Pro--L, whereas DBA/1 mice are low responders (10, 11). In order to determine whether the genetic control of immune response can be correlated with the number of antigen-sensitive precursor cells, spleen cell suspensions from normal and immunized SJL and DBA/1 donor mice were transplanted into lethally X-irradiated syngeneic recipients (incapable of immune response) along with (T, G)-Pro--L. Anti-(T, G)-Pro--L responses (donor-derived) were assayed in the sera of the hosts 12–16 days later. By transplanting graded and limiting numbers of spleen cells, inocula were found which contained one or a few antigen-sensitive precursors reactive with the immunogen. Using this method to estimate the relative numbers of such cells for the high responder SJL strain, one precursor was detected in ~1.3 x 10⁶ and ~7.2 x 10⁶ spleen cells from immunized and normal donors, respectively. In contrast, one precursor was detected in about 30 x 10⁶ spleen cells from low responder DBA/1 mice, irrespective of whether the donors had been immunized. These results indicate that the genetic control of immunity to the synthetic polypeptide antigen investigated is directly correlated to the relative number of precursor cells reactive with the immunogen in high and low responder strains.     

GENETIC CONTROL OF IMMUNE RESPONSES IN VITRO : II. CELLULAR REQUIREMENTS FOR THE DEVELOPMENT OF PRIMARY PLAQUE-FORMING CELL RESPONSES TO THE RANDOM TERPOLYMER L-GLUTAMIC ACID60-L-ALANINE30-L-TYROSINE10 (GAT) BY MOUSE SPLEEN CELLS IN VITRO

The cellular requirements for the development of primary IgG GAT-specific PFC responses in cultures of spleen cells from responder, C57Bl/6, mice stimulated with GAT and GAT-MBSA and in cultures of spleen cells from nonresponder, SJL and B10.S, mice stimulated with GAT-MBSA were investigated. Macrophages were required for development of responses to GAT and GAT-MBSA in cultures of spleen cells from responder mice and for responses to GAT-MBSA in cultures of spleen cells from nonresponder mice. Macrophages from nonresponder mice supported the development of responses to GAT by nonadherent responder spleen cells, indicating that the failure of nonresponder mice to respond to GAT is not due to a macrophage defect. Furthermore, responder macrophages supported the responses of nonadherent, nonresponder spleen cells to SRBC and GAT-MBSA, but not to GAT. This indicates that the capacity to respond to GAT is a function of the nonadherent population which is composed of thymus-derived (T) helper cells and precursors of antibody-producing cells. Treatment of spleen cells with anti-theta serum and complement before culture initiation abolished PFC responses to GAT and GAT-MBSA thus establishing the requirement for T cells in the development of PFC responses to these antigens. Since precursors of antibody-producing cells in nonresponder mice are capable of synthesizing antibody specific for GAT after stimulation with GAT-MBSA and since the response to GAT is thymus-dependent, it appears that nonresponder mice lack GAT-specific helper T cell function.     

IMMUNE RESPONSES IN VITRO : V. SUPPRESSION OFγM, γG,ANDγA PLAQUE-FORMING CELL RESPONSES IN CULTURES OF PRIMED MOUSE SPLEEN CELLS BY CLASS-SPECIFIC ANTIBODY TO MOUSE IMMUNOGLOBULINS

Suppression of Ig class-specific PFC responses by class-specific antibody to mouse immunoglobulin was studied in cultures of spleen cells from immunized mice. In contrast to cultures from normal mice where anti-µ suppressed responses in all Ig classes, anti-µ had progressively less suppressive effect on γ₁ and γ₂ responses in cultures from immunized mice with time after immunization. This was most pronounced at 10 days after immunization when anti-µ suppressed γM and γA responses, but had no or slight effect on γ₁ or γ₂ responses which were still suppressed with anti-γ₁ and anti-γ₂. These changes in precursor cell susceptibility to anti-µ were antigen specific.     

MECHANISMS OF GENETIC RESISTANCE TO FRIEND VIRUS LEUKEMIA IN MICE : I. ROLE OF89Sr-SENSITIVE EFFECTOR CELLS RESPONSIBLE FOR REJECTION OF BONE MARROW ALLOGRAFTS

Resistance to malignant erythropoiesis induced by Friend spleen focus-forming virus and resistance to marrow stem cell allografts are under genetic control. Strains of mice, e.g., C57BL/6 and B10.D2, which are homozygous for resistance at the Fv-2 locus, are also good rejectors of most bone marrow allografts. ⁸⁹Sr, a bone-seeking isotope, irradiates marrow but not other lymphoid organs and abrogates resistance to marrow allografts without suppressing T- or B-cell functions. Thus, marrow-dependent effector cells (M cells) seem to resist allogeneic stem cells. To test if the genetic resistance to Friend virus (FV) is also mediated by M cells, B6 mice were treated with ⁸⁹Sr using a dosage schedule known to abrogate resistance to allogeneic marrow cells. 9 days after FV infection of such mice, the spleens showed malignant erythroblastosis which could not be suppressed by prior hypertransfusion, a procedure which suppresses physiologic erythropoiesis. Such ⁸⁹Sr-treated B6 mice also supported extensive virus replication, while control mice did not. FV markedly suppressed the ability of ⁸⁹Sr-treated B6 mice to produce antisheep red blood cell (SRBC) antibodies, a feature seen normally only in genetically susceptible mice. Thus, ⁸⁹Sr-treated B6 mice behaved in these respects as if they were susceptible to FV. When increasing doses of ⁸⁹Sr were administered to B6 mice, a dose-related loss of resistance to FV was seen. Therefore, it appears that ⁸⁹Sr-sensitive M cells mediate the genetic resistance to FV. The results of experiments with ⁸⁹Sr indicated that genetically resistant mice would be expected to possess target cells which are susceptible to transformation by FV. To verify this corollary, bone marrow cells from B10.D2 (Fv-2^rr) mice were transplanted into previously infected and lethally irradiated DBA/2 (Fv-2^ss) recipients which share the same H-2^d alleles. 5–15 days later, the spleens of DBA/2 primary recipients yielded transformed cells which were capable of producing splenic tumor colonies upon transplantation into adult, unirradiated B10.D2 secondary recipients. Various control experiments clearly indicated that the tumor colonies so induced were of B10.D2 marrow origin. This indicated that B10.D2 stem cells could be transformed when allowed to interact with FV in the spleens of susceptible DBA/2 mice. However, 30 days after transplantation of B10.D2 bone marrow cells into DBA/2 recipients, no transformed cells were detected. Apparently, in the 30-day interval precursors in the B10.D2 marrow gave rise to mature M cells which resisted the leukemic process. Since M cells recognize hybrid or hemopoietic histocompatability antigens expressed on primitive normal and transformed hematopoietic cells, we suggest that M cells may exert surveillance by rejecting leukemic cells. Thus, marrow transplantation from genetically resistant donors may provide a new mode of treatment for leukemia, by providing precursors of M cells and other immunocompetent cell types.     

GENETIC CONTROL OF IMMUNE RESPONSES IN VITRO : IV. CONDITIONS FOR COOPERATIVE INTERACTIONS BETWEEN NONRESPONDER PARENTAL B CELLS AND PRIMED (RESPONDER x NONRESPONDER) F1 T CELLS IN THE DEVELOPMENT OF AN ANTIBODY RESPONSE UNDERIr GENE CONTROL IN VITRO

The conditions for cooperative interactions between nonresponder B10.S B cells and GAT-primed irradiated (C57BL/6 x SJL)F₁ T cells in the response by cultures of mouse spleen cells to GAT were investigated. GAT-specific antibody responses could be elicited by soluble GAT in cultures of GAT-primed irradiated (C57BL/6 x SJL)F₁ T cells with C57BL/6 B cells but not with B10.S B cells. In contrast, when GAT was presented to the cultures on F₁ macrophages or as aggregates of GAT with MBSA, GAT-specific PFC responses were observed with both B10.S or C57BL/6 B cells. Irradiated GAT-primed T cells were nevertheless essential for the development of these responses. The GAT-specific response of B10.S B cells in these cultures was inhibited by the addition of soluble GAT at culture initiation. These results indicate that genetic disparity at Ir loci is not an absolute barrier to T-B-cell cooperative interactions in the response to antigens under Ir gene control. The significance of these data for the function of Ir gene products in immunocompetent cells is discussed.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : VII. THE DEMONSTRATION, USING ALLOTYPIC MARKERS, OF ANTIBODY FORMATION BY IRRADIATION-RESISTANT CELLS OF IRRADIATED RABBITS INJECTED WITH NORMAL ALLOGENEIC BONE MARROW CELLS AND SHEEP ERYTHROCYTES

Bone marrow cells obtained from rabbits of one allotype were injected into irradiated rabbits of a different allotype. The recipients were also injected with sheep red blood cells, and their spleen cells were tested for plaque-forming capacity 7 days later. Spleen cells of all recipients gave large numbers of plaques as did spleen cells incubated with antiserum, directed toward donor allotype. However, incubation of the recipient spleen cells with antiserum directed toward recipient allotype completely suppressed plaque formation. These results demonstrate that antibody-formation in irradiated recipients of transferred lymphoid cells is a property of the recipient animal and that the antibody-forming cell is relatively irradiation-resistant. It was also demonstrated that only viable normal bone marrow cells are capable of transferring antibody-forming capacity to irradiated recipient rabbits. Neither sonicates nor heat-killed preparations of normal rabbit bone marrow cells possessed this capacity.     

GENETIC CONTROL OF IMMUNE RESPONSES IN VITRO : V. STIMULATION OF SUPPRESSOR T CELLS IN NONRESPONDER MICE BY THE TERPOLYMERL-GLUTAMIC ACID60-L-ALANINE30-L-TYROSINE10 (GAT)

In recent studies we have found that GAT not only fails to elicit a GAT-specific response in nonresponder mice but also specifically decreases the ability of nonresponder mice to develop a GAT-specific PFC response to a subsequent challenge with GAT bound to the immunogenic carrier, MBSA. Studies presented in this paper demonstrate that B cells from nonresponder, DBA/1 mice rendered unresponsive by GAT in vivo can respond in vitro to GAT-MBSA if exogenous, carrier-primed T cells are added to the cultures. The unresponsiveness was shown to be the result of impaired carrier-specific helper T-cell function in the spleen cells of GAT-primed mice. Spleen cells from GAT-primed mice specifically suppressed the GAT-specific PFC response of spleen cells from normal DBA/1 mice incubated with GAT-MBSA. This suppression was prevented by pretreatment of GAT-primed spleen cells with anti-θ serum plus C or X irradiation. Identification of the suppressor cells as T cells was confirmed by the demonstration that suppressor cells were confined to the fraction of the column-purified lymphocytes which contained θ-positive cells and a few non-Ig-bearing cells. The significance of these data to our understanding of Ir-gene regulation of the immune response is discussed.     

CELL INTERACTIONS BETWEEN HISTOINCOMPATIBLE T AND B LYMPHOCYTES : VI. COOPERATIVE RESPONSES BETWEEN LYMPHOCYTES DERIVED FROM MOUSE DONOR STRAINS DIFFERING AT GENES IN THE S AND D REGIONS OF THEH-2 COMPLEX

In our initial studies on the question of histocompatibility requirements in T-B-cell interactions, we found that no cooperation occurred with mixtures of T and B cells from BALB/c (H-2^d) and A/J (H-2^a) donors, respectively (1). These particular strains are identical for genes in the S and D regions of the H-2 complex but possess major differences at the K-end. Many differences are known to exist in the I region as well. Thus, these early data indicated that gene identities only at the D-end are insufficient to permit optimal cooperative interactions to occur under these conditions.     

GENETIC CONTROL OF THE ANTIBODY RESPONSE IN INBRED MICE : TRANSFER OF RESPONSE BY SPLEEN CELLS AND LINKAGE TO THE MAJOR HISTOCOMPATIBILITY (H-2) LOCUS

The transfer of spleen cells from (C3H x C57Bl/6) F₁ mice, capable of responding to (T,G)-A--L, into irradiated C3H parental recipients, normally incapable of responding to (T,G)-A--L, transfers the ability to make either a primary or secondary immune response to this synthetic polypeptide antigen. This localizes the genetic control of the ability to respond to the spleen cell population and indicates that the genetic control is exerted upon a process directly related to antibody formation. Studies with congenic strains of mice and linkage studies in segregating backcross populations show that the ability to respond to (T,G)-A--L and (H,G)-A--L is linked to the H-2 locus and can thus be localized to the IXth mouse linkage group. Note Added in Proof: Of the three possible recombinant animals noted in Tables IV and V, two were infertile. The third animal was not a recombinant, since progeny testing and reimmunization showed that this animal was an H-2²/H-2^k heterozygote capable of responding well to (T,G)-A--L.     

GENETIC CONTROL OF IMMUNE RESPONSES IN VITRO : III. TOLEROGENIC PROPERTIES OF THE TERPOLYMERL-GLUTAMIC ACID60-L-ALANINE30-L-TYROSINE10 (GAT) FOR SPLEEN CELLS FROM NONRESPONDER (H-2qANDH-2s) MICE

Although nonresponder, H-2^s and H-2^q, mice fail to develop GAT-specific PFC responses to GAT, they do develop GAT-specific PFC responses when stimulated by GAT complexed to an immunogenic carrier such as methylated bovine serum albumin. The studies described in this paper show that injection of nonresponder mice with GAT specifically decreases their ability to develop anti-GAT PFC responses to a subsequent challenge with GAT-MBSA. Addition of GAT to cultures of spleen cells from nonresponder mice also prevents development of the GAT-specific PFC responses stimulated by GAT-MBSA. Thus, interaction of nonresponder spleen cells with GAT leads to the induction of unresponsiveness in vivo and in vitro. Various parameters of the tolerance induction have been investigated and described. A comparison of the effects of GAT on B cells indicates that nonresponder B cells are more readily rendered unresponsive by soluble GAT than are responder B cells. The significance of these data for our understanding of Ir gene regulation of the immune response is discussed.     

IMMUNOLOGICAL MEMORY IN MICE : III. MEMORY TO HETEROLOGOUS ERYTHROCYTES IN BOTH T CELL AND B CELL POPULATIONS AND REQUIREMENT FOR T CELLS IN EXPRESSION OF B CELL MEMORY. EVIDENCE USING IMMUNOGLOBULIN ALLOTYPE AND MOUSE ALLOANTIGEN THETA MARKERS WITH CONGENIC MICE

Using anti-allotype sera and AKR anti θC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-θ serum and guinea pig serum (Anti θ + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti θ + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti θ + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.