GVHR elicited by products of class I or class II loci of the MHC: analysis of the response of mouse T lymphocytes to products of class I and class II loci of the MHC in correlation with GVHR-induced mortality, medullary aplasia, and enteropathy

A lethal graft-vs-host reaction (GVHR) was elicited by the injection into irradiated (700 rad) mice, reconstituted with T-depleted bone marrow cells (BM), of T lymphocytes incompatible for different loci of the major histocompatibility complex (MHC). The number of T cells needed to kill more than 50% of the recipients by day 40 was about 10(6) for GVHR elicited across the product of the K, D, or E locus, but about 10(5)--10--fold less-when the A locus was involved. The mortality was associated with a medullary aplasia in all strain combinations, but enteropathy was observed only in GVHR elicited by the products of class II, and not class I, loci. Mortality and medullary aplasia were diminished or absent in recipients reconstituted with BM cells from T cell donors instead of cells of the host genotype, which suggests a direct (cytolytic) T-hematopoietic cell interaction. Lymphoproliferation was evident within the host spleen and lymph node 5 days after injection of T lymphocytes incompatible for class II but not class I loci. Spleens from mice suffering from a lethal GVHR were examined by culture in limiting dilution to evaluate the frequency of anti-host T cells and to derive anti-host T cell clones and lines, whose properties were explored. In the GVHR elicited across the A or E region of the MHC, examined between days 7 and 19, a high frequency (10(-2] of anti-host cells was observed. The polyclonal cell lines isolated (16) all displayed MLR responsiveness, antigen-driven IL 2 production, and cytolysis for LPS blasts of the host genotype. However, among 13 clones isolated, two categories were observed: Lyt-2-, which were MLR responders and IL 2 producers (four of 13), and Lyt-2+, which were cytolytic but neither MLR responders nor IL 2 producers (nine of 13). In the GVHR elicited by the K or D region, examined between days 7 and 90, the frequency of anti-host cells was low (10(3) to 10(4], with a tendency to decrease during the progression of the disease. The lines (11) or clones (26) isolated from different mice were all Lyt-2+ and strongly cytolytic but proliferated poorly and produced no IL 2 in MLR. These findings suggest that the Lyt-2+ lymphocytes, recognizing the products of the class I loci, function in vivo without proliferation and without requiring helper T cells. Cell lines specific for class I or class II loci of the MHC produced interferon and colony-stimulating factors.     

Rapid changes in specificity within single clones of cytolytic effector cells

We find rapid changes in the specificity of the cytolytic effector cells in a mixed lymphocyte culture. The lysis patterns produced by cytolytic effector cells generated near limiting dilution in murine mixed lymphocyte reactions of three types, F₁ anti-parent (F₁(A × B) anti-A), allogeneic (C anti-F₁(A × B)), and F₁ anti-modified parent (F₁(A × B) anti-A-TNP), were investigated. Cultures were characterized by their ability or inability to lyse a panel of target cells (e.g., A, B, F₁). When individual cultures were tested at two different times, changes in lytic pattern were routinely seen, with some patterns reproducibly increasing in frequency and others reproducibly decreasing (e.g., patterns involving lysis of F₁ decreased in an F₁ anti-A response but increased in a C anti-F₁ response). X-linked isoenzyme analysis showed that changes can occur within a single clone of effector cells. These results imply that the T cell specificity repertoire continues to evolve during an ongoing immune response, a conclusion incompatible with clonal selection theory.     

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.     

Tissue distribution and polymorphism of minor histocompatibility antigens involved in GVHR

A graft-vs-host reaction (GVHR) develops after major histocompatibility complex (MHC)-compatible bone marrow-transplantation. In the genetic combination studied, B10.D2 donor cells differed from those of (DBA/2 x B10.D2)F₁ mice for multiple DBA/2 minor histocompatibility antigens (mHAg) and minor lymphocyte stimulating (M1s) antigens. We investigated the distribution and the cell type expression of mHAg in tissues that were potential GVHR targets, by means of specific T-cell clones derived from mice undergoing reaction. The T-cell clones studied had a CD4⁺ phenotype and recognized 12 distinct mHAg that were not be product of the Mls-1 ^a gene and that were presented predominantly in association with MHC class II A molecules. Our results indicate that DBA/2 alleles coding for mHAg are frequent in both laboratory and geographically unrelated wild mice. Each mHAg displays an individual pattern of expression on cells present in thymus, skin, gut, and liver. In addition, chimeric mice and established cell lines allowed the identification of cell types expressing mHAg. We found that most mHAg are present on lymphoid and monocyte-macrophage cells, whereas one, distinguished by its absence from lymphoid cells and damaged tissues, is expressed by monocyte-macrophage cells. Correspondence to: I. Miconnet.     

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.     

Cosegregation of the polymorphic C4 with the MHC in the frog,Xenopus laevis

Employing isoeletric focusing combined with enzyme-linked immunoelectrotransfer blot analysis, the fourth component of complement (C4) was analyzed in the two highly histocompatible, major histocompatibility complex homozygous groups (J and K) of Xenopus laevis. Each group had a characteristic C4 isoelectric focusing pattern, i. e., an isoelectric point range of 8.0–8.5 for J (C4 ^j C4 ^j ) and 7.6–8.1 for K (C4 ^k C4 ^k ). In (J x K)F₁ frogs, C4 proteins were expressed in a codominant fashion (C4 ^j C4 ^k ). In the backcrossed progeny B₁ [J × (J × K)F₁], those with C4 ^j C4 ^j rejected (J × K)F₁ skins hyperacutely (< 17 days), were high responders against (J × K)F₁ cells, and nonstimulators to J cells in mixed lymphocyte reaction (MLR), but they did not suffer from the graft-versus-host reaction (GVHR), even after the injection of (J x K)F₁ cell-stimulated J splenocytes. On the other hand, the B₁ frogs with C4 ^j C4 ^k acutely or chronically (> 17 days) rejected (J × K)F₁ skins, were low or nonresponders against (J × K)F₁ cells and high stimulators to J cells in MLR, and they suffered from GVHR after the injection of prestimulated J splenocytes. These results argue for the notion that the genes equivalent to mammalian class III map to the MHC at the phylogenetic level of the anuran amphibian.     

Clones of alloreactive T cells

We have developed a method which allows us to clone and reclone primed responder T cells derived from serially restimulated murine mixed lymphocyte cultures. We have derived clones from two such mixed lymphocyte cultures, A anti-B6 [A(B6)] and A anti-(B6XA)F₁ [A(B6A)]. In the A (B6) system, we have isolated clones which can be stimulated by B6 but not by (B6XA)F₁ cells. This implies the presence of a unique parental H-2^b MLR determinant which is absent on semi-allogeneic (B6XA)F₁ cells. In the A(B6A) system, we have isolated clones which can be stimulated by (B6XA)F₁ cells but not by B6 cells. This confirms our previous observation on the presence of unique hybrid MLR stimulating determinants on (B6XA)F₁ cells. Many of the “clones” derived primarily from soft agar seem to be contaminated and contain several different sets of primed responder cells with different reactivity patterns. Experiments in which we subcloned cells exhibiting selected reactivity patterns from such contaminated primary clones suggested that a T-cell growth factor or accessory cell is required for proliferation in soft agar following alloantigen recognition by primed responder cells.     

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.     

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     

Genetics of graft-versus-host reactions (GVHR)

Injection of 20×10⁶ donor lymph node cells (LNCs) into newborn allogeneic recipients incompatible with donors at theIC subregion of the mouseH-2 complex evoked both GVH splenomegaly and GVH mortality. The strength or severity of the allogeneic reactions induced varied as a function of the interallelic strain combination and was influenced particularly by properties of the recipientIC determinants. Thus,IC ^s determinants on recipient cells led to strong GVHR, whileIC ^d determinants induced a moderate GVHR, even when donors carrying differentIC alleles were used. However, responder donor genes also affected the degree of GVHR in some combinations. The effect on donor GVH potential of pre-exposing B10 donors to antirecipient antiserum (B10 anti-B10.A) was also studied. Spleen cells from B10 donors pre-exposed to alloantiserum for two to seven days exhibited a markedly reduced ability to cause GVH splenomegaly and GVH disease in newborn B10. A or (B10. A × B10) F₁ recipients. Inhibition of donor lymphocyte GVH potential waned eight to 14 days after antiserum pretreatment. Inhibition was shown to be specific for variousH-2 determinants and to be caused by antirecipient alloantibodies. Pre-exposure of donors to alloantiserum reduced the GVH potential of spleen cells but did not affect LNC reactivity.Ia antibodies and, to a lesser extent, anti-H-2D serum were shown to be able to inhibit GVHR. The results suggest that the observed reduction in donor GVH reactivity is caused by antibody-mediated central feedback inhibition. Anti-H-2 alloantibodies evidently play an important role in the network regulating allogeneic responses.     

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.     

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     

Interacting genetic factors controlling the antibody response against the H-2.2 specificity

The antibody response against the H-2.2 specificity has been studied in three H-2 ^d strains, B10.D2, DBA/2, and BALB/c, and their hybrids (B10.D2 × DBA/2)F₁ and (B10.D2 × BALB/c)F₁. The genetic control of the response appears to be complex: The three pure strains are responders, whereas both hybrids when immunized with C3H-HTG are nonresponders. Individual analysis of N3 offspring is compatible with the idea that, in this combination, an Ea-4 incompatibility between donor and immunized strain is necessary for the anti-H-2.2 response to occur. H-2 ^d /H-2 ^k hybrids (B10.BR × B10.D2)F₁ or (B10.BR × DBA/2)F₁ are responders when immunized with C57BL/10 (H-2 ^b ) but not with B10.A(2R) (H-2 ^h ), indicating that simultaneously recognized H-2 specificities are necessary for the anti-H-2.2 response.     

Tumour-induced altered gastrointestinal steady-states: absence of MHC restriction in the paraneoplastic gastrointestine

Abstract The growth of a number of experimental rodent tumours including the Lewis lung tumour (LLca) progressively compromises the integrity of the host's gastrointestine by inducing cytokinetic alterations in the small bowel resembling those generally defining the intestinal phase of a graft-versus-host reaction (GVHR). To determine whether the induction of this paraneoplastic gastrointestine (PGI) involves, similar to a GVHR, a disparity between the MHC of the donor (LLca tumour) and the recipient (host), PGI development was evaluated in various LLca tumour-bearing murine strains that were either ‘syngeneic’[C57BL/6 and BL/10 (H-2^b)], ‘semisyngeneic’[B6D2F₁ (H-2^bd) and B6C3F₁ (H-2^bk)] or ‘allogeneic’[C3H/HeJ (H-2^k) and DBA/2 (H-2^d)] to the H-2^b LLca tumour. The temporal appearance and magnitude of a PGI developing in either LLca-syngeneic or semi-syngeneic hosts, but not the allogeneic strains, suggested that the mechanism(s) involved in PGI development, like the GVHR, was restricted by the MHC. Subsequent studies using congenic strains [B10.A (H-2^k) and B10.D2/nSn (H-2^d)], however, demonstrated that the mechanism(s) responsible for the PGI was restricted by the non-MHC loci of the C57BL mouse. These observations were supported by the appearance of a LLca-induced PGI in various B10.A congenic strains carrying mutations at the I-A or I-E/I-J loci of the MHC. Not unlike the intestinal phase of a GVHR, development of the PGI required the participation of enhanced mucosal mast cells which were limited in the WCB6F₁ (S1/S1^d) but not the (+/+) murine strains. These observations are discussed in light of the postulated premature migration of immature thymocytes that accompany tumour growth and their ability to non-specifically enhance (or suppress) cell mediated immune reactions in the host.     

Genetic resistance of CBA and a mice to transplanted lymphoid and hemopoietic cells of CBA.M523 mutants and their F1 hybrids

(CBA × M523)F₁, (A × M523)F₁ and M523 lymphocytes grafted into lethally irradiated CBA or A mice temporarily lose their capacity to respond to test antigens (SRBC, Vi-antigenS. typhi). Immunoresponsiveness of F₁ cells is affected to a lesser degree in lethally irradiated M523 mice. Depression of response is absent in the CBA F₁ combination, in the syngeneic combination and in CBA mice which have received transplanted cells from F₁ hybrids which do not share theM523 mutation. The number of hemopoietic (CBA × M523)F₁ colonies was also reduced in CBA mice. Resistance of CBA mice to lymphoid (CBA × M523)F₁ cells develops 18 days after birth. It can be reduced by additional recipient preirradiation or preinoculation with (CBA × M523)F₁ spleen cells. The abrogated resistance can be partially restored by CBA spleen cells. The activity of (CBA × M523)F₁ lymphocytes passaged through CBA spleen is restored in syngeneic F₁ secondary recipients but inhibited again in the CBA secondary recipients. These results are consistent with the suggestion that resistance of lethally irradiated CBA mice to hemopoietic and lymphoid (CBA × M523)F₁ cells is mediated by immunologically competent, radioresistant recipient cells rapidly reacting to transplantation antigens coded by the mutantH-2K ^ka allele. These cells temporarily suppress the functional activity of transplanted cells but do not eliminate them.     

Expression of L3T4 molecules on Lyt-2+, class II-restricted antigen-specific T suppressor lymphocytes

Three bovine serum albumin-specific Lyt-2⁺ T suppressor (Ts) cell clones from CBA/J mice have been analyzed with regard to expression of L3T4 molecules. All three Ts-cell clones can be stained with monoclonal antibodies (mAb) to L3T4. Tested for the two clones restricted to recognition of E^k determinants, antigen-specific proliferation on antigen-presenting cells, but not the proliferation induced by conditioned medium can be inhibited by L314-specific mAb. In a similar way, Ts-cell cytolytic effector functions can be blocked by L3T4-specific mAb. Thus L3T4 structures seem to play a role in Ts-cell functions. Furthermore, the data support the view that L3T4 expression can be a property of class II-restricted T cells irrespective of their Lyt phenotype.     

Activation or suppression of bactericidal activity of macrophages during a graft-versus-host reaction againstI-A andI-J-region differences,respectively

Systemic graft-versus-host reactions (GVHR) were induced in F₁ heterozygous mice by injecting 10⁸ parental lymphocytes. The Anti-Thy 1.2-sensitive, T-cell mediated activation of macrophages was assessed by their increased capacity to destroy a facultative intracellular bacteriumListeria monocytogenes. The difference inMHC regions causing a GVHR that induced high levels of macrophage activation mapped toI-A. In contrast, differences atK orD, in any of the otherH-2 subregions or in the non-H-2 background, includingMls alone or in combination, did not induce a GVHR leading to macrophage activation, unless these differences were combined with a difference atI-A. The numbers of parental cells needed to activate macrophages via a GVHR caused byI-A vs. non-I-A differences, varied at least 30- to 100-fold. When parental cells were injected into F₁ offspring of parents differing atI-J, growth ofListeria was enhanced significantly; this negative effect on macrophages was not seen when parental combinations differing atI-A alone were compared with those differing atI-A plusI-J orI-J plus otherH-2 regions.     

Control of the antibody response of C57BL/6 Lyt-congenic strains to the Lyt-3.1 alloantigen by a gene linked to theLyt-2 locus

Congenic anti-Lyt-3.1 sera have recently been produced by immunizing B6-Lyt-2^a mice with thymocytes from either B6-Lyt-2^a, Lyt-3^a or B6-Lyt-2^a, Lyt-3^a, H-2^k mice (Boos et al. 1978). Surprisingly, mice of the congenic strain B6 failed to produce either anti-Lyt-2.1 or anti-Lyt-3.1 cytotoxic antibodies after identical immunizations. To determine the genetic basis for the difference in response to Lyt-3.1, (B6 × B6-Lyt-2^a)F_a mice and progeny of the backcross, (B6 × B6-Lyt-2^a)F₁ × B6-Lyt-2^a, were immunized with B6-Lyt-2^a, Lyt-3^a, H-2^k thymocytes. In addition, thymic biopsies of backcross progeny were performed and thymocytes tested for the Lyt-2.2 antigenic specificity. Results indicate that gene(s) governing the immune response to Lyt-3.1 is (are) linked to theLyt-2 locus, and that the responder allele (linked toLyt-2 ^a ) shows very poor penetrance in Lyt-2^a/Lyt-2^b mice.     

T-cell subsets in the thyroids of mice developing autoimmune thyroiditis

To examine the role of T-cell subsets in the development of thyroid lesions, female CBA/J mice were immunized with 60 μg mouse thyroglobulin (MTg) in 0.1 ml complete Freund's adjuvant in both hind footpads. The thyroids were removed 12–21 days later, pooled, and dispersed. The cell suspension was examined by membrane immunofluorescence for the distribution of Thy-1⁺, Lyt-1⁺, Lyt-2⁺, and sIg⁺ lymphocytes. For comparison, peripheral blood leukocytes (PBL) from the same animals were similarly examined. Throughout this 10-day interval, B cells in the thyroid were consistently below 5%, whereas B cells represented 19–24% of PBL. Thy-1⁺ cells in PBL ranged from 45 to 59%, whereas Thy-1⁺ cells in the thyroid were 37–50%. However, only thyroidal T cells showed a consistent decline with time and were replaced gradually by cells without T or B cell markers. In particular, there was a clear shift in the Lyt-1⁺:Lyt-2⁺ ratio from about 7 down to 2 in the thyroid as the early predominance of Lyt-1⁺ cells was followed by a relative increase in Lyt-2⁺ cells. Our results show that there is an accumulation of Lyt-1⁺ and Lyt-2⁺ cells in the infiltrated thyroid. These cells may include MTg-reactive, helper, and cytotoxic T cells which localize (or differentiate) in the thyroid and initiate the lesions.     

Tcrb-V3+3T-cell deletion and a new mouse mammary tumor provirus,Mtv-44

Genes encoding endogenous superantigens causing Tcrb-V3⁺ T-cell deletion co-segregate with mouse mammary tumor proviruses (Mtv),Mtv-3, Mtv-6, andMtv-13. In additionMtv-1 has been implicated in deletion of these T cells. We have examined levels of Tcrb-V3⁺ T cells andMtv integrations in the following off-spring and their parental strains, [(CBA-T6 × NZW)F₁ × CBA], [(CBA × C3H/He)F₁ × CBA], and [(B10.S (9R) × NOD]F₁ mice. We show that a newMtv (Mtv-44) from NZW mice andMtv-1 from C3H/He mice cosegregate with genes encoding ligands for partial deletion of Tcrb-V3^s+ T cells and that some NOD mice have an additionalMtv (Mtv-45) which is closely linked toMtv-3. Address correspondence and offprint requests to: K. Tomonari.