Clonal analysis of the T lymphocytes involved in parent versus Fn1 graft-versus-host reaction

A systemic graft-versus-host reaction (GVHR) leading to 50% mortality by day 20 was elicited by the injection of CBA (10⁵) or B10 (10⁶) parental T lymphocytes into irradiated (750 rad) and bone marrow protected (CBA x B10)F₁ recipients. Between days 12 and 28 the spleens of the sick mice were analyzed by limiting dilution, performed with irradiated F₁ cells and a source of interleukin-2 (IL-2), to determine the frequency of cells with an antihost proliferative or cytolytic activity and to derive T lymphocyte clones. The frequency of cells with antihost proliferative or cytolytic activity was approximately 10^–3 in either combination. In the CBA vs F₁ GVHR, all eight clones isolated with anti-F₁ activity were Lyt-2^–, noncytolytic, mixed lymphocyte reaction (MLR) responders and IL-2 producers, three of which mapped to the A ^b locus, while in the B10 anti-F1 combination, eight of the nine anti-F₁ clones isolated were Lyt-2⁺, poor MLR responders and non-IL-2 producers, but cytolytic and mapping to K _k . These findings suggest a much higher frequency of T cells recognizing the A-locus antigens in the CBA than in the B10 strain.     

Immunosuppressive activity of murine newborn spleen cells. I. Selective inhibition of in vitro lymphocyte activation.

Cell-mediated immune responses to newborn lymphocyte alloantigens were investiated using mitogen activation, mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). Spleen cells from 1- to 5-day-old (C57BL/6 × Balb/c) F₁ mice co-cultured with maternal strain (BALB/c) splenocytes did not affect DNA synthesis of maternal strain cells in the presence of concanavalin A or phytohemagglutinin. Newborn cells did inhibit the lipopolysaccharide response of maternal strain lymphocytes and these cells also depressed DNA synthesis when added to MLR cultures of BALB/c and C57BL/6 spleen cells. Newborn cells expressed poor stimulatory capacity in semiallogeneic MLR and also caused marked inhibition of DNA synthesis when added to semiallogeneic MLR containing BALB/c (responder) and CB6F₁ adult splenocytes (stimulator). The suppression of MLR by neonatal cells persisted for the first 2 weeks of life and was associated with a soluble factor released during culture. The suppressive activity was almost completely abrogated after depleting the T-cells from newborn splenocytes. However, these same cells did not interfere with the in vitro generation of cytotoxic lymphocytes in the CML assay. The selective immunosuppressive properties of newborn spleen cells may be important during pregancy by protecting the immunologically alien fetus from rejection by the mother.     

Influence of the H-2 u haplotype on immune function in F1 hybrid mice

Recently we reported that antigen-primed T cells from (H-2 ^u × H-2 ^s)F₁ and (H-2 ^u × H-2 ^q)F₁ mice responded poorly in vitro to antigen in the context of antigen-presenting cells of the non-H-2 ^u parent. It was suggested that this effect might be due to unbalanced expression of parental antigens in the F₁ hybrid with the result that the non-H-2^u A antigens were greatly reduced or absent in these mice. If this were the case, non-H-2^u Ia-A cells might be expected to stimulate a mixed lymphocyte reaction (MLR) when cultured with Fl responder cells. When tested, (SJL × PL)F₁ responder cells reacted strongly to SJL stimulator cells. There was no significant reaction to PL stimulator cells. The use of major histocompatibility complex (MHC) congenic mice showed the stimulatory antigens to be associated with the MHC. The MLR could be blocked significantly by monoclonal A-specific antibody of the appropriate specificity. When a monoclonal antibody reactivewith a private epitope associated with A^s was used to probe for the presence of A^s on the surface of (SJL × PL)F₁ spleen cells, no antigen could be detected, indicating loss or alteration of this antigen. These findings suggest that an alteration of the expression of the parental A^s molecule may be responsible for this phenomenon.Abbreviations used in this paper APC antigen-presenting cells - BSS balanced salt solution - CTL cytotoxic T lymphocyte - IL-2 interleukin-2 - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - T₂ suppressor T lymphocyte     

Xid-defective male (CBA/N x C57BL/6)F1 accessory cells present bovine insulin to long-term cultured F1-restricted T-cells

The reactivity of H-2^b-restricted murine T cells towards bovine insulin was reported to depend on the expression of Ia.W39, a private specificity of I-A^b, on antigen-presenting cells. Cells of male (CBA/N x B6)F₁ mice carrying the mutation xid on the X chromosome lack Ia.W39 on the cell surface. These cells are unable to present bovine insulin to primed T cells derived from female (CBA/N x B6)F₁ mice. We show here that spleen cells of male (CBA/N x B6)F₁ hybrids served perfectly as accessory cells for the insulin-dependent induction of a proliferative response of long-term cultured T cells with (B10 x B10.BR)F₁ genotype, restricted to recognizing insulin in the context of F₁-unique I-A determinants. The epitope on the insulin molecule essential for stimulation was determined to depend on the glutamic acid residue in position 4 of the A chain of insulin. This contrasts with the H-2^b-restricted response of B6 mice to bovine insulin, which appears to be directed at the A chain loop determinant (amino acids A8 and A10). These data suggest that distinct I-A^b-encoded structures, the expression of which is regulated independently, may serve as components of restriction elements for H-2^b and (H-2^b x H-2^k)F₁ restricted T cells, which are specific for different epitopes of bovine insulin.     

Quantitation of the T cell deficiency in RFM mice with host versus graft disease.

Host versus graft (HVG) syndrome is the fatal complex of lesions which has been observed in six inbred strains of mice following the perinatal inoculation of related F₁ hybrid spleen cells. Morphological studies have indicated that the key lesion is the depletion of peripheral T lymphocytes due to inflammatory destruction and failure of the thymus to replace them. In the present studies, tests of T-cell function were done on RFM mice, which had developed HVG disease following perinatal inoculations of (T₆ × RFM)F₁ spleen cells. As compared to control values, HVG spleen cell suspensions showed loss of reactivity to phytohemagglutinin (PHA) = 90%, to concanavalin A (Con A) = 94%, to (T₆ × RFM)F, cells in the mixed lymphocyte reaction (MLR) = 82%, to DBA cells in MLR = 94%, and to DBA mastocytoma cells in cell-mediated lympholysis (CML) = 95%. Lymph node cell suspensions showed losses of reactivity to PHA = 83%, to Con A = 62%, to (T₆ × RFM)F₁ cells in the MLR = 91%, and to DBA cells in the MLR = 77%. The CML activity of nodal cells to DBA mastocytoma cells varied widely from 12 to 273% of control values, and averaged 121%. Filtration of HVG spleen cells through nylon fiber columns failed to restore low responses to PHA to normal values. This suggested that the macrophage-like, adherent accessory cells were not acting as suppressors of T-cell responses in HVG disease. The deficits in all T-cell-mediated functions tested so far, appeared to correlate very well with quantitative morphological studies which showed the loss of 98% of the small lymphocytes normally present in the thymic dependent portions of the splenic white pulp. It is suggested that experimental HVG disease may serve as a model for immunodeficiency syndromes of the Nezelof type which are also characterized by T-cell deficiency, poor primary antibody responses, and the presence of variable amounts of serum immunoglobulins.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

Haplotype preference in lymphocyte differentiation. II. F1 hybrid helper T cells generated with antigen-bearing parental macrophages can cooperate with B lymphocytes of either parent.

Carrier-specific helper T cells were generated in F₁ hybrid mice by either conventional immunization procedures or by repeated immunizations with antigen-bearing macrophages derived from either F₁ or parental donors. The F₁ helper T cells generated in these various ways were then analyzed for their capacities to help hapten-primed B lymphocytes derived from each of the two parental strains as well as from F₁ donors in the development of secondary anti-hapten antibody responses. These analyses were conducted using two different types of in vivo assay systems as well as a totally in vitro system. Under all circumstances, helper T cells from F₁ mice, primed either in conventional fashion or with antigen bearing parental or F₁ macrophages, were capable of interacting effectively with B lymphocytes of each parent and of F₁ origin. Moreover, in the case of F₁ helper cells primed with antigen-bearing parental macrophages, there was no evidence of preferential helper activity for parental B lymphocytes corresponding to the type of macrophage used for sensitization; this was true irrespective of whether in vivo or in vitro assay systems were employed. The relevance of these findings and others which are either similar to, or discordant with, them to the general question of genetic restrictions in macrophage-T lymphocyte interactions is discussed.     

Selective suppression of the murine autologous mixed lymphocyte reaction by physiological concentrations of hydrocortisone: effects on cell-surface Ia antigens

Strong, adult (Type II) autologous mixed lymphocyte reactions (AMLR) were observed in cultures of lymphoid cells from both A.TH and A.TL mice. These were suppressed by more than 90% in the continuous presence of 7.5 × 10^?8M hydrocortisone-21-sodium succinate. This concentration of hormone had minimal effects on the allogeneic mixed lymphocyte response (MLR) and the mitogenic response to concanavalin A (Con A). Higher concentrations suppressed all three responses. Treatment of autologous cell mixtures for the first 30 hr with 7.5 × 10^?8M hydrocortisone resulted in a 78% suppression of the AMLR. This was not associated with a detectable decrease in the quantity of Ia antigens on the stimulator-cell surface, as evaluated by the susceptibility of treated cells to antibody dependent, complement-mediated lysis, using [A.TH × B.10M]F₁ anti-A.TL antiserum. Hence, this suppression did not appear to result from an alteration of the antigens putatively associated with stimulation of the AMLR. Separate pretreatment of stimulator and responder cells with 7.5 × 10^?8M hydrocortisone followed by culturing with appropriate companion cells had no major effect on the AMLR. Therefore, low-dose hydrocortisone did not appear to selectively eliminate or permanently inactivate subpopulations of responder or stimulator cells. Rather, it appeared to regulate active cellular processes that are initiated by the coculturing of these cells and are required for the early stages of autologous lymphocyte activation.     

H-Y antigen: No evidence for alleles in wild strains of mice

H-Y antigen(s) coded or controlled by the Y chromosome in a variety of wild mouse strains have been compared with those of the inbred laboratory strains C57BL/6 (B6) and C57BL/10 (B10). H-Y antigen(s) were detected by H-2-restricted cytotoxic T cells from B6 and B10 female mice primed in vivo and boosted in vitro with syngeneic male spleen cells: There was no difference in the degree of H-Y specific lysis of male cells from the C57BL strains and of F₁ hybrids or B6 congenic mice carrying the Y chromosome from the wild mouse strains examined. This result indicated that at the level of target cell specificity the H-Y antigen(s) from wild and laboratory strains were indistinguishable. H-Y antigen(s) were also found to be indistinguishable at the level of the in vitro induction of the anti H-Y cytotoxic response: F₁ female mice, primed in vivo and boosted in vitro with homologous F₁ male cells, all made H-Y-specific responses and where it could be examined, the target cell specificity of the anti-H-Y cytotoxic cells showed that B10 male cells as well as the homologous F₁ male cells (where the Y chromosome was derived from the wild strain) were good targets. Finally, possible differences in H-Y transplantation antigens between the wild strains and the B10 laboratory strain were examined by grafting F₁ male mice, the progeny of B10 females, and wild strain males with B10 male skin. These grafts were not rejected during an observation period of more than 9 months. Taken together, neither the cytotoxic data nor the skin graft data provide any evidence for allelism of H-Y even though the mouse strains examined were collected from widely disparate geographical locations.     

Histocompatibility-2 system in wild mice

Six semicongenic lines carrying differentt haplotypes on the background of strain C57BL/10Sn (B10.t strains) and a (B10 ×T/t ⁰) F₁ hybrid were tested against one another in the mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML) assays. In every instance, the MLR results paralleled those of the CML typing: strain combinations giving a positive result in one assay gave a positive result in the second; combinations in which no response was observed in the MLR assay also failed to kill target cells specifically in the CML assay. Furthermore, the MLR and CML results were concordant with the results of the serological typing of these strains, as reported previously by us. The combined results suggest sharing ofH-2 hyplotypes between B10.t¹² and B10.t³², between B10.t⁶ and B10.t^w1, and between B10.t^w2 and (B10. ×T/t ⁰) F₁. These data support the conclusion, reached in our previous publication, that members of the samet-complementation group, with few exceptions, shareH-2 haplotypes.     

Requirement for the location of both appropriate and irrelevant H-2 antigens on the same stimulator cell for unspecific DNA-synthesis inhibition by the H-2-antigen-primed,specific suppressor T cells

Specific suppressor T cells (SSTC), primed in vivo with H-2 antigens, have been shown previously to inhibit DNA synthesis in the one-way, three-cell mixed lymphocyte reaction (MLR) provided that (a) the stimulator cells bear the priming H-2 antigens, and (b) the responder cells possessIC+S regions homologous to those of the SSTC. Anti-B10.A BlO.A(2R) SSTC (anti-D^d) and anti-A.AL A.TL SSTC (anti-K^k) are shown here to be able to inhibit the DNA synthesis triggered in MLR, not only by the corresponding antigens, D^d and K^k, respectively, but also by irrelevant, third-party H-2 and Mls products provided that the corresponding and third-party antigens are presented on the same stimulator cell. If stimulatorH-2 regions, whose products interact with SSTC and responders, are located on different stimulator cells within the particular MLR, SSTC activity is not elicited. Participation of cytotoxic T lymphocytes in DNA-synthesis suppression is ruled out. Direct contact or location of the inhibited responder cell very close to SSTC is considered to be required for the development of SSTC activity.     

Studies of murine lymphocytes and alloantigens: I. Ly-6 mitogen and MLR responses

Antisera to the mouse lymphocyte surface alloantigens Ly-6.1 and Ly-6.2 were used to further study the functional distribution of these antigens. After selective depletion with antiserum + rabbit complement (RC), lymph node or spleen cells from Ly-6 congenic (C3H and C3H.B6-Ly-6^b) and noncongenic strains of mice were tested for: (a) their proliferative responses to T- and B-cell mitogens; and (b) their proliferative responses to alloantigens, or ability to stimulate in the MLR. Lymphoid cells required in the proliferative responses to the mitogens leucoagglutinin, concanavalin A (Con A), lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were Ly-6⁺. Lymph node responder cells in the mixed lymphocyte reaction (MLR) were also Ly-6⁺, whereas spleen stimulator cells were Ly-6^?. Treatment of lymph node cells with anti-Ly-6 sera in the absence of RC had no specific blocking effect on the response to any of these mitogens. The studies indicate that the Ly-6 antigen is a potentially valuable marker for distinguishing between functionally distinct Ly-1⁺ T-cell subsets.     

Functional requirements of (Phe,G)-A--L-specific T-cell clones of (H-2 b x H-2 kF1 origin

T-cell clones specific for the synthetic polypeptide antigen poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) of (C57BL/6 x C3H/HeJ)F₁ origin were tested for their biological activities. One group of clones was restricted in its proliferative response to the H-2 ^b haplotype, the second to the H-2 ^k haplotype, and the third to the F₁ unique Ia determinants. All the clones which proliferated in response to antigen secreted interleukin-2 (IL-2) following stimulation. The H-2 restriction of the IL-2 secretion was the same as that of the proliferation. Two of the clones tested, C.6 and C.10, could provide help to B cells in antibody production. However, the genetic restriction profile of the helper activity was less stringent than that for the proliferative response. Thus, C.6, which proliferated in the presence of F₁ antigen-presenting cells only, could help B cells and accessory cells of C3H/HeJ. C.10, which was restricted in its proliferative response to the H-2 ^b haplotype, could collaborate with B cells and accessory cells of the H-2 ^k haplotype as well. The antibody response of both clones was restricted to the parental or F₁ strains.Abbreviations used in this paper (T, G)-A-L poly-(LTyr, LGlu)poly(DLAla)--poly(LLys) - (Phe, G)-A--L poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) - APC antigen-presenting cells - Con A concanavalin A - FCS fetal calf serum - IL-2 interleukin-2     

CML typing of serologically identicalH-2 mutants

Cell-mediated lympholysis (CML) reactions were studied among four strains of C57BL/6 (B6) mice carrying mutant alleles (H-2 ^ba ,H-2 ^bd ,H-2 ^bg , andH-2 ^bh ) at thez1 locus in theK end ofH-2 ^b and the original B6 (H-2 ^b ) strain. Cross killing of target cells from lines that had not participated in the mixed lymphocyte reaction (MLR) was extensive, but usually less intense than that of target cells of stimulator cell genotype. The extent of CML crossreactivity could be limited by using cells from F₁ hybrid mice as responders in MLR. In a comprehensive analysis of the cytotoxicity exerted by 20 MLR combinations with homozygous, and 10 MLR combinations with F₁ hybrid responder cells, 19 different CML cytotoxicity patterns were identified, corresponding to at least 19 different CML target specificites. When the number of CML mismatches of each mutant with the originalH-2 ^b was calculated,H-2 ^ba was found to be most distinct fromH-2 ^b ,H-2 ^bs andH-2 ^bd were closest toH-2 ^b , andH-2 ^bh occupied an intermediate position. The validity of this sequence of relatedness is supported by published reports on skin graft survival times and on the interaction of T lymphocytes with virus-infected target cells using cells fromz1 locus mutants.     

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due toH-2I-region differences. In both cases the MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. —Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in restimulation during MLR-II. — These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.Abbreviations used in this paper Lad lymphocyte-activating determinant - MLR mixed lymphocyte reaction - MLR-I, MLR-II primary, secondary MLR - PRC primed responder cells - LCT dye exclusion lymphocytotoxicity microtechnique - RR relative response     

Unsuitability of the assay for cell-mediated lympholysis in inbred mice for H-Y antigen determination of human cells

Summary This study examined the H-Y-specific in vitro restimulation of splenocytes from in vivo intraperitoneally (i.p.) primed C57B1/6 (B6) female mice. In vivo priming was carried out with human male or female fibroblasts or peripheral blood lymphocytes, respectively. It was attempted to measure the in vitro H-Y-specific activity by cell-mediated lympholysis and by cell proliferation. ³[H]Thymidine incorporation was determined in mixed lymphocyte cultures (MLCs) of xenogenetic primed female splenocytes (responder cells) and of syngeneic lethally irradiated male splenocytes (stimulator cells). The xenogenic H-Y presentation by in vivo sensitization did not induce in the in vitro restimulation system an H-Y-specific cell proliferation or in the effector phase the generation of H-Y-specific killer cells. The assay for celimediated lympholysis and lymphocyte proliferation after xenogeneic priming and syngeneic in vitro restimulation is, thus, not suitable for H-Y testing of human cells.     

Alteration in lymphocyte recognition repertoire during aging: II. Changes in the expressed T-cell receptor repertoire in aged mice and the persistence of that change after transplantation to a new differentiative environment

Changes in the T-lymphocyte alloreceptor repertoire associated with aging by exploring the frequency of cytotoxic T-lymphocyte precursors (CTLp) available for activation by various major histocompatibility complex (MHC) haplotypes in mice of different ages have been investigated. There was no consistent pattern of change in CTLp frequencies. Thus, for instance, while the frequency of responder C57Bl/6 CTLp for ATH alloantigen decreased with age, the frequency for C3H alloantigen increased. There was no significant change in the overall frequency of splenic CTLp (assessed irrespective of antigen specificity). No evidence was found that CTL produced by activated CTLp of aged mice were less specific in their lytic capacity that CTL produced by CTLp of young mice. However, by assaying responder CTLp cultures at limiting dilution we obtained evidence that the “burst size” (mean lytic capacity per responder well assayed at limiting dilution) was diminished with age of the donor of the CTLp pool. Furthermore, we obtained evidence that the apparent affinity of CTL for their target antigen was consistently decreased when those effector cells were derived from a pool of CTLp of aged mice. All of these changes reflected in mature T cells derived from aged mice were already apparent in the bone marrow stem cell pool of aged individuals and were not due to environmental influences alone, as assessed by the phenotype of T cells derived from young or old bone marrow stem cells transplanted to young or aged recipient mice. A final study has examined evidence for more subtle changes in the T-cell alloreceptor repertoire, reflecting heterogeneity in young or aged mice in the recognition repertoire associated with a given antigenic specificity. By preparing F₁ anti (parent anti-F₁)-suppressor cells directed against CTL from young parental mice (a, b, c), or aged parental mice (x, y, z), we have explored the heterogeneity in the anti-C3H alloreceptor repertoire in individual young or aged C57Bl/6 mice. Suppression by immunized F₁ animals was assessed in tissue culture (inhibition of mixed lymphocyte culture (MLC) responses) or in vivo (inhibition of lethal GvHD induced by inoculation of parental lymphocytes into sublethally irradiated F₁ hybrid mice). Irrespective of the assay system used, the data suggests that the receptor repertoire of aged T lymphocytes uses recognition structures different from those of young individuals, and that there is less individual-to-individual variation in the receptor repertoire of aged mice than in young mice.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Murine T-cell hybridomas that produce lymphokine with macrophage-activating factor activity as a constitutive product

Responder spleen cells primed to alloantigens in vivo could generate high degree of cytotoxicity against low- or nonimmunogenic stimulators such as thymocytes or uv light-treated spleen cells in vitro. However, a removal of adherent cells from primed responder cells remarkably reduced the cytotoxicity after stimulation with such low-immunogenic stimulators. Adding a small number of peritoneal adherent cells (PACs) also suppressed the cytotoxic activity of unseparated responders against low-immunogenic stimulators. These suppressive effects by PACs were blocked by indomethacin. By adding prostaglandin E₂, cytotoxic T lymphocyte (CTL) generation of primed unseparated responders against low-immunogenic stimulators was suppressed; however, cytotoxic activity against mitomycin C-treated stimulators was not suppressed. These results suggested that prostaglandins released from PACs selectively inhibited the function of splenic adherent cells that were required for CTL generation of primed responder spleen cells against low-immunogenic stimulators in vitro.     

Secondary mouse mixed lymphocyte reaction (MLR) in vitro: Correlation between primed cells reactivity and serological typing for Ia specificities

B10.M (H-2^f) spleen lymphocytes were cultured for 14 days with X-irradiated B10.P (H-2 ^p ) lymphocytes. These primed cells were then tested for their capacity to respond to a secondary stimulation induced by a panel of mouse cells carrying differentH-2 haplotypes. The third-party cells induced various degrees of proliferation which could be expressed as a percentage of the proliferation induced by the primary stimulating strain (relative response = RR) and could then be classified according to the RR. Anti-Ia antibodies present in a B10.M anti-B10.P serum were studied by the dye exclusion lymphocytotoxicity technique (LCT) against the same panel, after absorption of H-2K, D antibodies on B10.P platelets. The strain panel could then be classified according to the LCT titers of the absorbed immune serum. A significant correlation (r=0.96,P<0.01) was found between both classifications. According to the Ia chart, the public specificities involved were Ia.6, 7, and 13, but these did not fully explain either the primed cell reactivity or the LCT results. An unexpected crossreactivity was observed between B10.P and B10. Absorption-elution experiments with A.TH anti-A.TL serum demonstrated that B10 and B10.P share the Ia.3 antigen. These results indicate that the structure(s) recognized by the primed lymphocytes is the Ia antigen(s).Abbreviations used in this paper RR Relative response - LCT Dye exclusion lymphocytotoxicity technique - MLR Mixed lymphocyte reaction - PC Primed cell - MHC Major histocompatibility complex - PLT Primed lymphocytes typing test