Biomedical subjects
Publications and source records attributed to J K Inman.
Another procedure for preparing dinitrophenyl (DNP) sheep erythrocytes (SRBC) using the DNP-alanylglycylglycyl hapten has been developed. These DNP-SRBC also are stable for as long as 1 month, but the conjugation procedure is much simpler to carry out than the previously reported method.
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CBA/N mice harbor an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. Hapten-specific plaque-forming cell (PFC) responses to haptenated proteins by B cell-defective CBA/N mice and CBA/N X C3H/HeN F1 male mice can be blockaded by concomitant exposure to polysaccharide agents bearing the same hapten. Various experimental approaches were explored in an attempt to study this phenomenon in a syngeneic cell transfer system. Unprimed donor spleen cells were unable to mount adequate PFC responses to dinitrophenylated-hemocyanin (DNP-KLH) in irradiated, syngeneic recipients. DNP-KLH-primed donor spleen cells produced strong PFC responses after challenge in irradiated recipients, and these secondary responses were subject to hapten-specific blockade by DNP-derivatized polysaccharide agents. Both direct and indirect PFC responses could be blocked. DNP conjugated to bovine serum albumin also produced hapten-specific blockade in this cell transfer system. Under some cell transfer conditions normal CBA/N X C3H/HeN F1 female spleen cells were just as susceptible to hapten-specific blockade as defective F1 male spleen cells.
Mice were immunized i.c. with various enlarged haptens conjugated to bovine serum albumin and mixed with the cationic, surface active lipid, dimethyl dioctadecyl ammonium bromide (DDA). This immunization generated delayed-type hypersensitivity (DH) in these mice without detectable concomitant antibody formation. The DH was measured as footpad swelling. However, both direct and indirect hapten-specific PFC could be detected in peripheral lymph nodes and in spleens 4 days after a challenge injection. Although the adjuvant, DDA, promotes a strong cross-reactivity in DH between heterologous hapten-carrier complexes, the antibody-forming cells produced 4 days after challenge showed relatively high specificity for the immunizing hapten. This indicates only weak cross-reactivity at the antibody-forming cell level co-existent with high cross-reactivity in DH expression to the same hapten-carrier complexes. These results are consistent with the possibility that B-cell receptors may be capable of expressing a greater degree of hapten specificity than T-cell receptors. It is tentatively concluded that Thelper cells participating in antibody formation may represent a subset of the T cells involved in DH.
A method is described for preparing derivatives of alkali-stable polysaccharides for coupling to immunogen carriers or to sheep red blood cells (SRBC) for use in hemagglutination (HA) and plaque-forming cell (PFC) assays. Inulin, a beta (2 leads to 1)-linked polyfructosan was partially derivatized with carboxyl, aminoethyl or (p-aminophenyl)butyryl groups; the latter derivative was coupled to SRBC following diazotization. Optimal conditions for the sensitization of SRBC with inulin were given. The immunological reactivity of the inulin molecule was unaffected by the derivatization reactions, and high, reproducible anti-inulin HA titers for inulin-binding myeloma proteins were found using these specifically sensitized SRBC. The sensitized SRBC were stable for assays for over 2 weeks. Problems with spontaneous agglutination or distortion of sensitized SRBC, normally seen in other procedures, e.g., methods using stearoyl-inulin, were not encountered.
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CBA/N mice, a mutant CBA subline, harbor an X-linked B-cell defect which prevents them from mounting immune responses to certain thymic-independent antigens such as pneumococcal polysaccharides and haptenated-Ficoll derivatives. These mice and the hybrid male progeny of CBA/N females are also exquisitely sensitive to a hapten-specific blockade of their otherwise adequate immune responses to thymic-dependent antigens such as N-2,4-dinitrophenylated-hemocyanin (DNP-KLH). As little as 10 ng of a DNP-Ficoll conjugate given 2 h before immunization with a 5,000-fold greater dosage of DNP-KLH, virtually abolishes the 4th-day direct plaque-forming cell (PFC) response specific for DNP. Responding hybrid (CBA/N x C3H/HeN) female mice are resistant to such blockade even at DNP-Ficoll dosages increased by three orders of magnitude. The DNP hapten and Ficoll must be chemically joined for this blocking effect to occur, and increasing the hapten derivatization of Ficoll increases its blockade-invoking capacity. Significant blockade can be produced by administering DNP-Ficoll as early as 4 days before or as late as 4 h after immunization with DNP-KLH. All currently available data point to the defective B cell as the target of this hapten-polysaccharide-mediated blockade. Mice bearing B memory cells, however, are refractory to such blockade. In addition, DNP-Ficoll injections which cause virtually total blockade of 4th-day primary direct PFC responses to DNP-KLH have little or no effect on the development of DNP-reactive B-cell memory measured at either 8 or 30 days. These findings suggest very different blockade susceptibilities for B cells or their precursors at various stages of differentiative development. Our findings also lead to the formulation of testable hypotheses regarding the mechanism of this selective B-cell blockade phenomenon.
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Both nude mice (nu/nu) and their heterozygous littermates (nu/+) were injected with a single IP dose of 300 mg cyclophosphamide (CY)/kg. CY is a known immunosuppressive agent, which affects primarily B lymphocytes. Immunization with the thymus independent antigen DNP-AGG59-Ficoll after CY treatment disclosed that restoration of the primary direct PFC response occurred more rapidly in nude mice than in nu/+ mice. However in these same experiments, the primary indirect PFC response, recovered earlier in nu/+ mice than in nude mice. After CY treatment, secondary indirect PFC responses were delayed in both nude and nu/+ mice, but the greatest effect was seen in nude mice. The data suggest that the presence of T cells has little if any influence on the recovery capacity of those B cells which are destined to become direct PFC. However the recovery of B cells which are destined to produce indirect PFC responses is facilitated by the presence of T cells.
Delayed-type hypersensitivity (DH) in the mouse was provoked with different hapten-carrier complexes mixed with the cationic, surface-active lipid, dimethyl dioctadecyl ammonium bromide (DDA). DH was measured as footpad swelling. Conjugates of bovine serum albumin (BSA) with the small haptens dinitrophenyl (DNP), 'arsonate' (ARS) and 'sulphonate' (SULPH) served to generate strong DH reactions towards the homologous antigen. Insertion of a tripeptide spacer between the hapten and carrier resulted in lower DH reactivity. Optimal dosages and optimal time intervals between sensitization and DH elicitation were determined for the enlarged hapten-carrier complexes. Cyclophosphamide (CY) treatment, before priming with complexes mixed with DDA, caused a 5-6 day delay in the expression of DH but failed to evoke enhanced DH for any of the antigens tested. A broad array of cross reactions between small and enlarged hapten-carrier complexes showed a relative lack of specificity in these DH responses. The results are compared with others reported in the literature and are explained mainly by the effects of electrostatically bound lipid groups of DDA in the sensitizing conjugates.
Mice with the CBA/N defect are unresponsive to the hapten phosphorylcholine (PC) even when presented on a variety of immunogenic carriers. Since these mice have the variable region gene for PC, their inability to respond may reflect deletion or suppression of the line of B lymphocytes which is responsible for the anti-PC response.
Immunization of rabbits or mice with a single, chemically defined hapten elicits populations of plaque-forming cells (PFC) detectable not only on sheep erythrocytes (SRBC) bearing the immunizing hapten, but also on SRBC bearing structural analogues of the immunizing hapten. Most of these analogue-reactive PFC preferentially lyse analogue-conjugated SRBC and cannot be detected on erythrocytes bearing the immunizing hapten. Thus, they represent heretofore largely unstudied components of the secretory B-cell response to haptenic immunization, and they have been termed alloreactive PFC. Such alloreactive PFC are detectable using either classical small haptens or tripeptide-enlarged counterparts of these classical haptens. They are present in large numbers both in direct and in indirect PFC assays, and they are elicited in response to both thymic-dependent and thymic-independent antigens. Relatively few alloreactive PFC can be attributed to cells producing hapten-carrier or "bridge area"-specific antibodies. Since the antibodies released by alloreactive PFC can also be detected by passive hemagglutination, their presence does not appear attributable to vagaries of complement activation. Numerous coexisting alloreactive PFC populations are detectable after haptenic immunization. In early direct PFC responses it is not nucommon for a single alloreactive PFC population to outnumber the population of PFC detectable on SRBC bearing the actual immunizing hapten. These alloreactive PFC may be the source of at least some of the new "nonspecific" Ig which is formed at the time of immunization but about which little is known for lack of available techniques. Some possible implications of these findings on the specificity of B precursor cell activation are discussed.
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T cells from guinea pigs immunized with the hapten 2,4-dinitrophenyl (DNP)-coupled directly to mycobacteria are of interest since they recognize and respond to DNP conjugated to many but not all carriers. The experiments reported here further analyze the structure of the complex, chemically defined antigenic determinants recognized by such T cells. These antigenic determinants can have DNP coupled either to the xi-amino group of lysyl residues or to the hydroxyl group of tyrosyl residues. Furthermore, essential contributions to the determinant recognized by such T cells are made by amino acid residues to which the hapten is not attached. Such residues are thought to be close to the hapten group itself, since introducing a small spacer between hapten and carrier prevents recognition. The hapten itself is also recognized and discriminated from other haptens with great precision by these T lymphocytes. The strain of guinea pig immunized affects the precise specificity characteristics of the responding T cells, in a way that may reflect the activity of histocompatibility-linked immune response genes. Finally, the characteristics of the immunogen have been studied. It is thought that the lipid content of the mycobacteria may be critical in inducing the hapten-reactive T cells, and this is supported by finding similar responses in T cells from guinea pigs immunized with DNP protein to which lipid has been covalently attached. Thus, the T-cell population being studied, while recognizing haptens with great precision, appears to require a larger determinant for activation than do hapten-specific B lymphocytes.
The specificity of C57BL/10 cytotoxic effector cells generated by in vitro sensitization with autologous spleen cells modified with a series of related nitrophenyl compounds was investigated. The failure of trinitrophenyl (TNP)-sensitized effector cells to lyse TNP-beta-alanylglycylglycyl(AGG)-modified target cells is presented as evidence contradicting the intimacy or dual receptor model or T-cell recognition in its simplest form. Data are also shown indicating that sensitization with N-(3-nitro-4-hydroxy-5-iodophenylacetyl)-AGG-modified stimulating cells generates noncross-reacting clones of cytotoxic effector cells.
Splenic lymphocytes from four C57BL/10 congenic mouse strains were sensitized in vitro to N(-3-nitro-4-hydroxy-5-iodophenylacetyl)-beta-alanylglycylglycyl-(N) modified autologous lymphocytes. The effector cells generated after 5 days of culture were assayed on a series of either N-modified phytohemagglutinin-stimulated spleen cells or N-modified tumor cells. The results indicated in all cases that both N modification of the targets and H-2 homology between the modified stimulating and target cells are required for lysis to occur. In each case the effector cells were found to lyse N-modified target cells only when there was homology at either or both ends of the major histocompatibility complex (MHC) between the stimulator and target cells. B10.BR lysed targets sharing alleles at K (or K plus I-A) and/or at D. B10.A effector cell specificity was mapped to K (or K plus I-A) and/or the D half of the MHC (D or D plus I-C and/or S). The two regions of specificity determined for B10.D2 effector cells were D (or D plus S plus I-C) and a region not including D of the MHC. C57BL/10 effector cells lysed N-modified targets only if there was target cell H-2 homology at K, I-A, and I-B or at the D serological region. As in the trinitrophenyl (TNP) system (6) B10.BR and B10.A effector cells lysed targets sharing K end H-2 serological regions greater than target cells sharing D-end serological regions. The C57BL/10 effector cells were shown to react to the K end greater than the D end, which differed from the equal reactivity seen in the TNP system for this strain. The data are consistent with the hypothesis that the antigen recognized by the effector cell includes an altered H-2 serological cell surface product. That the reaction is not "hapten specific" and the H-2 homology is required only for effector:target cell interaction was excluded by the use of two F1 combinations in which lysis of only N-modified target cells sharing the H-2 haplotype with the stimulating parental strain was obtained. Finally, it was demonstrated that N and TNP modification create distinct new antigenic determinants, since an effector cell sensitized to one modifying agent will lyse only H-2 matched target modified with that same modifying agent.