Selective engraftment of memory CD4+ T cells with an unusual recirculation pattern and a diverse T cell receptor-Vβ repertoire into scid mice

Young (H-2^d, L^d+) severe combined immunodeficiency (scid) mice were injected intravenously with 10⁵ CD4+CD8^? T cells purified from spleen, thymus or lymph nodes (LN) of dm2 (H-2^d, L^d ) donor mice. In the immunodeficient recipients, the lymphoid compartment in the splenic white pulp was repopulated with donor-type T cells and cellularity in the red pulp was increased. In addition, donor-type CD4⁺ T cells repopulated the peritoneal cavity, mesenteric LN and the lamina propria of the small intestine of scid mice, but were undetectable in thymus and peripheral (inguinal, axillary) LN. Histological examination of repopulated mesenteric LN showed expanded subcapsular sinuses, repopulated cortical areas, but poorly developed high endothelial venules (HEV) indicating deficient blood-LN lymphocyte recirculation. The engrafted CD4⁺ T cell population had the surface phenotype of memory T cells (CD44/Pgp-l^high CD45RB^low) and expressed the Peyer's patch HEV-specific homing receptor CD49d (LPAM-1), but not the LN HEV-specific homing receptor LECAM-l. The CD4+ T cell population in spleen and mesenteric LN of transplanted scid mice displayed a diverse T cell receptor-V(3 repertoire. Transfer of titrated numbers (10³, 10⁴, 10⁵ cells per mouse) of CD4⁺ T cells into scid mice established donor-type T cell populations with this unusual homing pattern in all recipients. Repeated serial transfers of dm2 CD4⁺ T cells through young scid mice revealed an extensive in vivo expansion potential of transferred cells for > 18 months. The experimental system described represents an in vivo model to study the functional competence and the differentiation potential of a murine memory CD4⁺ T cell subset.     

Gut-homing CD4+ T cell receptor αβ+ T cells in the pathogenesis of murine inflammatory bowel disease

We studied which T cell subsets from the gut-associated lymphoid tissue (GALT) can migrate out of the gut mucosa and repopulate GALT compartments of an immunodeficient (semi)syngeneic host. Many distinct lymphocyte subsets were found in GALT of immunocompetent H-2^d (BALB/c, BALB/c^dm2, C. B-17+/+) mice. No antigen receptor-expressing lymphoid cells were found in GALT of congenic C. B-17 scid/scid (scid) mice. The heterotopic transplantation of a full-thickness gut wall graft from the ileum or colon of immunocompetent (C. B-17+/+, BALB/c^dm2) donor mice onto immunodeficient scid mice selectively reconstituted a CD3⁺ T cell receptor αβ⁺ CD4⁺ T cell subset. CD4⁺ cells of this subset expressed the surface phenotype of mucosa-seeking, memory T cells. In the immunodeficient scid host, this gut-derived CD4⁺ T cell subset was found in spleen, peritoneal cavity, mesenteric lymph nodes (LN), epithelial layer and lamina propria of the small and large intestine, but not in peripheral LN. Scid mice heterotopically transplanted with gut from a congenic, immunocompetent donor developed clinical and histological signs of inflammatory bowel disease (IBD). Hence, the selective repopulation of GALT compartments with CD4⁺ T cells from normal GALT plays an essential role in the pathogenesis of IBD in an immunodeficient host.     

Distinct roles for lymphotoxin-α and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue

Specialized roles for the pro-inflammatory cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) were characterized in TNF/LTα^?/? and TNF^?/? mice established by direct gene targeting of C57BL/6 ES cells. The requirement for LT early in lymphoid tissue organogenesis is shown to be distinct from the more subtle and varied role of TNF in promoting correct microarchitectural organization of leukocytes in LN and spleen. Development of normal Peyer's patch (PP) structure, in contrast, is substantially dependent on TNF. Only mice lacking LT exhibit retarded B cell maturation in vivo and serum immunoglobulin deficiencies. A temporal hierarchy in lymphoid tissue development can now be defined, with LT being an essential participant in general lymphoid tissue organogenesis, developmentally preceeding TNF that has a more varied and subtle role in promotion of correct spatial organization of leukocytes in LN and spleen. PP development in TNF^?/? mice is unusual, indicating that TNF is a more critical participant for this structure than it is for other lymphoid tissues.     

Adoptive Transfer of Low Numbers of CD4+ T Cells into SCID Mice chronically treated with Soluble IL-4 Receptor does not prevent Engraftment of IL-4-Producing T Cells

After intravenous injection of 10⁵ purified, lymph node (LN)-derived dm2 (H-2^d/L^d) CD4⁺ T cells into young C.B-17 scidjscid (severe combined immunodeficiency, SCID) mice (H-2^d/L^d+), the transplanted L^d- T cells show a selective pattern of engraftment: they repopulate the spleen, the lamina propria of the small intestine and the mesenteric LN (but not other peripheral LN) of the immunodeficient host. CD4⁺ cells repopulating different lymphoid organs of the SCID recipient mice produce interleukin-2 (IL-2) and interleukin-4 (IL-4) in response to polyclonal stimulation in vitro. Some evidence has recently been provided that cytokines (e.g. IL-4) present at the site of antigen stimulation in vivo decisively influence the pattern of cytokines expressed by T cells activated at these sites. We therefore asked if neutralization of IL-4 by chronic treatment of SCID mice with high doses of recombinant soluble IL-4 receptor (sIL-4R) changes the IL-4 or IL-2 expression pattern of CD4⁺ T cells adoptively transferred into young SCID recipients. Transplanted SCID mice were chronically treated with two different, recombinant murine sIL-4R proteins. The experimental series further included groups of transplanted SCID mice treated with a recombinant human sIL-4R protein (which does not bind murine IL-4), treated with the anti-murine IL-4 monoclonal antibody (MoAb) 11B11, or non-treated. Transplanted SCID mice treated with the recombinant murine sIL-4R protein preparations displayed detectable sIL-4R serum levels, which demonstrates that the substitution therapy could maintain neutralizing serum levels of anti-IL-4 activity in SCID mice. By contrast, no serum sIL-4R levels were detectable in the sensitive ELISA readout in transplanted SCID mice which were non-treated, treated with the MoAb 11B11, or treated with the recombinant human sIL-4R protein. The efficiency and the pattern of CD4⁺ T-cell engraftment, and the lymphokine-producing phenotype of the engrafted dm2 CD4⁺ cells, was not affected by the continuous IL-4-neutralizing treatment of mice with either the MoAb 11B11 or the soluble IL-4R preparations. Hence, in contrast to the published evidence of the dramatic effect of IL-4 on the lymphokine-producing phenotype of CD4⁺ T cells stimulated in vitro or in vivo, the chronic suppression in vivo of IL-4 activity (by either different sIL4-R protein constructs, or by the anti-IL-4 MoAb 11B11) did not lead to preferential engraftment of Th1-type CD4⁺ T cells after adoptive transfer of CD4⁺ T-cell populations into an immunodeficient recipient.     

CD3+ T Cells in Severe Combined Immunodeficiency (SCID) Mice. V. Allogeneic T Cells Engrafted into SCID Mice Do Not Induce Graft-Versus-Host Disease in spite of the Absence of Host Veto and Natural Suppressor Cell Activity

Intravenous injection of 10(6) to 10(7) non-fractionated spleen cells (SC) from C57BL/6 (B6, H-2b) mice into completely allogeneic, immunodeficient H-2d severe combined immuno deficiency (scid) mice leads to engraftment of allogeneic donor T cells. Mice analysed in the tenth week posttransfer had engrafted donor-type CD4+ and CD8+ T cells in the spleens but showed no clinical evidence of graft-versus-host disease (GVHD). Transfer of allogeneic T cells engrafted in scid recipients did not induce GVHD upon i.v. injection into secondary scid recipients and lead in most recipients to engraftment of a pure CD4+ T-cell population. Experiments were carried out to investigate the reason(s) for the lack of GVHD in recipient scid mice, i.e. the presence of allotolerance in the engrafted donor T cells. Scid spleen cells (SC) efficiently stimulated alloreactive responses of B6 T cells: scid SC stimulated H-2d-specific cytotoxic responses in a B6 anti-scid mixed lymphocyte culture in vitro, and scid SC injected i.v. into B6 mice efficiently primed splenic cytotoxic lymphocyte precursors against H-2d alloantigens. Moreover when assayed in vitro, no veto activity or natural suppressor activity was detectable in scid SC. These data demonstrate that tolerizing mechanisms currently believed to operate in vivo can not explain the fact that allogeneic T cells injected i.v. into immunodeficient scid mice become tolerized against host-type alloantigens. Our results are discussed in the light of clinical experience of allogeneic T-cell transfer in scid infants.     

CD3+ T cells in severe combined immunodeficiency (scid) mice. IV. Graft-vs.-host resistance of H-2d scid mice to intravenous injection of allogeneic H-2b (C57BL/6) spleen cells.

Intraperitoneal injection of 2 x 10(7) nonfractionated spleen cells (SC) from C57BL/6 (B6, H-2b) mice into completely allogeneic immunodeficient H-2d scid mice induced clinical and histological signs of acute graft-vs.-host disease (GVHD), with all transplanted severe combined immunodeficiency (scid) mice dying in the 3rd week post-transfer. In contrast four out of five scid mice survived for greater than 7 weeks after intravenous (i.v.) injections of equal numbers of B6 SC. Intravenously allotransplanted scid mice analyzed in the 8th week post-transfer had engrafted donor-type CD4+ and CD8+ T cells in the spleens but showed no clinical or histological evidence of GVHD. i.v. injection of 10(7) or 10(6)O B6 SC engrafted allogeneic T cells in spleens of scid recipients; in contrast, i.v. injection of 10(5) nonfractionated B6 SC or 3 x 10(5) cell sorter-purified, naive or anti-H-2d-primed splenic CD4+ or CD8+ B6 T cells led to rejection by young scid recipient mice. B6 T cells engrafted into spleens of scid mice after i.v. injection showed proliferative anti-host alloreactivity in vitro. No cytotoxic reactivity against host-type alloantigens was found in standard 4-h 51Cr-release assays. These data demonstrate that allogeneic T cells injected i.v. into immunodeficient scid mice are partially tolerized against host-type alloantigens.     

T cell specialization at environmental interfaces: T cells from the lung and the female genital tract of lpr and gld mice differ from their splenic and lymph node counterparts

Mice homozygous for lpr and gld accumulate CD4^?CD8^? (double-negative, DN) B220⁺CD5¹⁰Thy-1¹⁰ αβ T cells in the spleen and lymph nodes (LN), while mucosal gut T cells are normal. To study other mucosa-associated T cell populations, we examined T cell subsets separated according to expression of αβ T cell receptor, CD4, CD5, CD8, Thy-1 and B220 in the lung and the female genital tract (FGT) of adult MRL lpr, C3H lpr and C3H gld mice. αβ T cell accumulation was detected in both the FGT and the lungs of lpr and gld mice but, in contrast to the spleen and LN, equal proportions of DN B220⁺ and CD4⁺ of CD8⁺ (single-positive, SP) B220^? T cells were observed in these sites, and the T cells had an increased expression of Thy-1 and CD5. Staining for CD44, L-selectin, and CD45RB revealed a higher percentage of effector/memory T cells in lpr and gld lungs and FGT compared to spleens and LN. CD69 expression suggested chronic activation of DN and SP T cells in lpr and gld lungs and FGT. Thus, we show that FGT and lung resident T cells are affected by lpr and gld mutations, but that their phenotypes are distinct from those of systemic T cells. These data suggest that T cells associated with FGT and lung mucosal tissues represent a separate lineage from systemic T cells, and/or that the abnormal T cells in lpr and gld mice are selected against in mucosal surfaces exposed to environmental antigen.     

Immunological memory function of the T and B cell types: distribution over mouse spleen and lymph nodes

Spleens from LAF₁ mice injected intravenously with sheep erythrocytes (SE) are relatively rich in memory T cells early in the immune response (1 to 3 days) and rich in memory B cells as the response progresses (2 weeks or more). Marked cooperation for the secondary immune response in vitro was obtained by combining 10⁶ spleen cells from LAF₁ mice, taken 2 days after intravenous priming with SE, with 10⁷ spleen cells from day 14 primed mice. The results indicate relative deficiencies in the spleen for B memory cells on days 1 to 2 and for T memory cells on day 14 after priming. Day – 14, but not day – 2, immune lymph node (LN) cells could replace the day – 2 spleen cells (anti-Thy 1.2 sensitive) in the in vitro cooperation with day – 14 immune spleen cells. Immune spleen cells taken 4 to 7 days after priming contain more equivalent numbers of B and T memory cells, but 10 to 7 days after transfer of such immune spleen cells without SE into irradiated recipients the T memory cells were again more prominent in lymph node and the B memory cells in spleen as shown by in vitro cooperation studies. These results suggest that during the second week after intravenous injection of SE relatively more T than B memory cells migrate from spleen to lymph node, resulting in an imbalance in the splenic memory cell population favoring B memory cell function.     

Non-H-2-associated genetic regulation of cytotoxic responses to hapten-modified syngeneic cells Effect on the magnitude of secondary response and helper T cell generation after in vivo priming

The present study investigates the role of non-H-2 genes in controlling generation of the H-2-restricted, T cell-mediated cytotoxic response against trinitrophenyl (TNP)-modified syngeneic cells (TNP-self). Spleen cells from C3H/He (H-2^k) or B10.BR (H-2^k) normal mice or from mice primed to TNP in vivo by skin painting with trinitro-chlorobenzene were used (a) for in vitro sensitization to TNP-self and (b) as a source of radioresistant helper cells for augmenting the TNP-self cytotoxic T lymphocyte (CTL) response generated by normal syngeneic spleen cells. Although spleen cells from unprimed mice from these two strains exhibited a comparable CTL response in a primary culture, a strong difference was observed in a secondary CTL response after in vivo priming. CTL activities generated in the secondary culture were much stronger in C3H/He than in B10.BR strains. This difference in the magnitude of secondary CTL responses was paralleled by generation of strong and weak helper cell activity in C3H/He and B10.BR, respectively. No detectable difference was observed between the two H-2^k strains in the lysability of target cells and ability of stimulating cells to activate the primed unirradiated cells and radioresistant helper cells. This genetic difference detected in the H-2^k haplotype was also demonstrated in the H-2^b haplotype, by using C3H.SW and C57BL/10 mice which bear non-H-2 background genes corresponding to C3H/He and B10.BR mice, respectively. These results indicate the existence of a control mechanism influenced by non-H-2 genes, in addition to the established H-2-linked gene control.     

H - 2 ANTIGENS OF CERTAIN t AND NON-t MICE REACT WITH THE SAME MONOCLONAL ANTIBODY

Monoclonal antibody 212.i.4.2 mediated complement-dependent lysis of spleen and lymph node cells carrying the t^w1, t^w12, t^w71, t⁶, t^w73, and t^Lub1 haplotypes, while cells from mice carrying 11 other t haplotypes were not lysed. The antibody also detected an epitope controlled by genes in the H-2D^d region of non-t mice. A molecule of 46,000 molecular weight was immunoprecipitated by 212.i.4.2 from detergent extracts of ¹²⁵I-labelled spleen cells of +/t^w12 and B10.D2 mice. The H-2^dm2 mutation did not alter the expression of the epitope recognized by 212.i.4.2. However, the H-2^dm1 mutation decreased the reactivity of lymphoid cells with the antibody in cytotoxicity tests, and 212.i.4.2 immunoprecipitated little or no protein from extracts of B10.D2(R106) spleen cells which carry the H-2^dm1 mutation.     

CD25+ Treg specifically suppress auto‐Ab generation against pancreatic tissue autoantigens

To study B‐cell tolerance against non‐lymphoid tissue autoantigens, we generated transgenic rat insulin promoter (RIP)‐OVA/hen egg lysozyme (HEL) mice expressing the model antigens, OVA and HEL, in pancreatic islets. Their vaccination with OVA or HEL induced far less auto‐Ab titers compared with non‐transgenic controls. Depletion of CD25⁺ cells during immunization completely restored auto‐Ab production, but did not affect antibodies against a foreign control antigen. Depletion at later time‐points was not effective. OVA‐specific CD25⁺ FoxP3⁺ T_reg were more frequent in the autoantigen‐draining pancreatic LN than in other secondary lymphatics of RIP‐OVA/HEL mice. Consistently, B cells were suppressed in that LN and also in the spleen, which is known to concentrate circulating antigen, such as the antigens used for vaccination. Suppression involved preventing expansion of autoreactive B cells in response to autoantigen, reducing antibody production per B‐cell and isotype changes. These findings demonstrate that CD25⁺ T_reg suppress auto‐Ab production against non‐lymphoid tissue antigens in an antigen‐specific manner.     

Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies

Acute infection with Trypanosoma cruzi, the aetiological agent of Chagas’ disease, results in parasitaemia and polyclonal lymphocyte activation. It has been reported that polyclonal B‐cell activation is associated with hypergammaglobulinaemia and delayed parasite‐specific antibody response. In the present study we analysed the development of a B‐cell response within the different microenvironments of the spleen during acute T. cruzi infection. We observed massive germinal centre (GC) and extrafollicular (EF) responses at the peak of infection. However, the EF foci were evident since day 3 post‐infection (p.i.), and, early in the infection, they mainly provided IgM. The EF foci response reached its peak at 11 days p.i. and extended from the red pulp into the periarteriolar lymphatic sheath. The GCs were detected from day 8 p.i. At the peak of parasitaemia, CD138⁺ B220⁺ plasma cells in EF foci, red pulp and T‐cell zone expressed IgM and all the IgG isotypes. Instead of the substantial B‐cell response, most of the antibodies produced by splenic cells did not target the parasite, and parasite‐specific IgG isotypes could be detected in sera only after 18 days p.i. We also observed that the bone marrow of infected mice presented a strong reduction in CD138⁺ B220⁺ cells compared with that of normal mice. Hence, in acute infection with T. cruzi, the spleen appears to be the most important lymphoid organ that lodges plasma cells and the main producer of antibodies. The development of a B‐cell response during T. cruzi infection shows features that are particular to T. cruzi and other protozoan infection but different to other infections or immunization with model antigens.     

Ir gene expression on T cells: Effect of a monoclonal antibody directed against I region-controlled determinants on T cells

A monoclonal antibody directed at an I region-controlled epitope uniquely expressed on T cells (Iat) was studied for its in vivo effect on the antibody response under the control of an Ir gene. The antibody was produced by a hybridoma made from A.TH spleen cells immune to A.TL (anti-I^k), that was selected for its reactivity with T but not B cells and macrophages, and thus was designated as anti-Iat^k. The injection of this anti-Iat^k into H-2^k, H-2^b and H-2^k×bF₁ mice resulted in the suppression of antibody response to poly-L-(His,Glu)-poly-D,L -Ala–poly-L -Lys [(H,G)-A–L] in H-2^k and F₁ mice but not that to poly-L -(Tyr,Glu)-poly-D,L -Ala–poly-L -Lys [(T,G)-A–L] both in H-2^b and F₁ mice. The adoptive cell transfer of the combinations of anti-Iat^k-or normal mouse serum-treated T and B cells into irradiated hosts demonstrated that anti-Iat^k primarily affected (H,G)-A–L-specific helper T cells but not B cells and macrophages, resulting in the specific elimination of the antibody response. Suppressor T cells were not induced by the treatment with anti-Iat^k. The antibody specifically eliminated the (H,G)-A–L-specific but not (T,G)-A–L-specific helper T cells in F₁ spleen cells that had been primed with both (H,G)-A–L and (T,G)-A–L. The results indicated that anti-Iat^k affected the H-2^k-associated Ir gene function born by T cells but not by antigen-presenting cells, which was expressed on F₁ helper T cells with apparent exclusion of the other allele, and implied that the Iat antigen on helper T cells is one of the sites of expression of Ir genes.     

Cytotoxic T cell response against lymphoblasts infected with Moloney (Abelson) murine leukemia virus. Methodological aspects and H-2 requirements

A method for infection of lymphocytes with Moloney(Abelson) murine leukemia virus [M(A)-MuLV] is described. Only lymphoblasts obtained after stimulation of normal spleen cells by the B cell mitogen lipopolysaccharide (LPS) were satisfactory targets for virus-specific, secondary cytotoxic T lymphocytes (CTL), whereas spleen cells stimulated by the T cell mitogen concanavalin A were not. The secondary CTL response against M(A)-MuLV could be efficiently measured using M(A)-MulV-infected LPS blasts as stimulating cells for secondary in vitro restimulation and as target cells for virus-specific destruction. Cold target inhibition demonstrated virus specificity of CTL. The T cell character of the cytotoxic cells was demonstrated by their sensitivity to anti-Thy-1.2 treatment. Using syngeneic virus-infected LPS blasts as target and stimulator, CTL responses were measured with effector cells from C57BL mice of the H-2^b haplotype and of recombinant haplotypes sharing either K or D alleles with H-2^b. In analogy with previous studies on Moloney virus-specific CTL. it was observed that C57BL/6 (H-2^b) effector cells predominantly lysed D^b-compatible, virus-infected target cells; B10.A(5R), (K^bD^d) effector cells showed a poor CTL response against syngeneic, virus-infected target cells. The combined findings indicate the existence of an Ir gene in the H-2D region regulating the CTL response against Moloney leukemia virus.     

Vβ11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in mice bearing H-2q versus H-2b haplotypes

We have recently demonstrated that toxic shock syndrome toxin-1 (TSST-1) expanded Vβ11⁺ T lymphocytes contribute to Staphylococcus aureus arthritis and sepsis-induced mortality. Interestingly, Vβ11⁺ T-cell mediated joint pathology varies in different mouse strains. In this study, we characterized the in vitro pattern of Vβ11⁺ T-cell expansion by TSST-1 in mice with various genetic backgrounds. Mice expressing major histocompatibility complex (MHC) class II I-E molecules did not expand Vβ11⁺ T cells upon stimulation with TSST-1. Using B10 congeneic I-E negative mouse strains, we found that the TSST-1-expanded Vβ11⁺ T cells in B10Q (H-2^q) and B10M (H-2^f) mice but not in B10B (H-2^b) mice. Antigen-presenting cells (APC) from B10Q mice, L cells and lymphoma cell line transfected with a q gene did not restore the deficient Vβ11⁺ T-cell expansion by TSST-1 in purified T cells from B10B mice. In contrast, I-A^b APC were able to stimulate Vβ11⁺ T cells from H-2^q mice. Furthermore, Vβ11⁺ T cells in H-2^b mice did expand when exposed to staphylococcal enterotoxin A (SEA). These findings suggest that the T-cell repertoire, skewed by clonal deletion and inactivation of self-reactive T cells, accounts for the different magnitude of Vβ11⁺ T-cell expansion among the different mouse strains.     

Co-expression of CD8α in CD4+ T cell receptor αβ+ T cells migrating into the murine small intestine epithelial layer

We investigated the surface phenotype of CD3⁺CD4⁺ T cell receptor (TCR) αβ⁺ T cells repopulating the intestinal lymphoid tissues of C.B-17 scidlscid (severe-combined immunodeficient; scid) (H-2^d, L^d+) mice. CD4⁺ CD8^? T cells were cell sorter-purified from various secondary and tertiary lymphoid organs of congenic C.B-17 +/+ (H-2^d, L^d+) or semi-syngeneic dm2 (H-2^d, L^d?) immunocompetent donor mice. After transfer of 10⁵ cells into young scid mice, a mucosa-homing, memory CD44^hi CD45RB^lo CD4⁺ T cell population was selectively engrafted. Large numbers of single-positive (SP) CD3⁺ CD2⁺ CD28⁺ CD4⁺ CD^8? T cells that expressed the α₄ integrin chain CD49d were found in the spleen, the mesenteric lymph nodes, the peritoneal cavity and the gut lamina propria of transplanted scid mice. Unexpectedly, large populations of donor-type doublepositive (DP) CD4⁺ CD8α⁺ CD8β^? T cells with high expression of the CD3/TCR complex appeared in the epithelial layer of the small intestine of transplanted scid mice. In contrast to SP CD4⁺ T cells, the intraepithelial DP T cells showed low expression of the CD2 and the CD28 co-stimulator molecules, and of the α₄ integrin chain CD49d, but expressed high levels of the α_IEL integrin chain CD103. The TCR-Vβ repertoire of DP but not SP intraepithelial CD4⁺ T cells was biased towards usage of the Vβ6 and Vβ8 viable domains. Highly purified populations of SP and DP CD4⁺ T cell populations from the small intestine epithelial layer of transplanted scid mice had different abilities to repopulate secondary scid recipient mice: SP CD4⁺ T cells repopulated various lymphoid tissues of the immunodeficient host, while intraepithelial DP CD4⁺ T cells did not. Hence, a subset of CD3⁺ CD4⁺ TCRαβ⁺ T cells apparently undergoes striking phenotypic changes when it enters the microenvironment of the small intestine epithelial layer.     

The Effect of Antigen Stimulation on the Migration of Mature T Cells from the Peripheral Lymphoid Tissues to the Thymus

Although the maturation and export of T cells from the thymus has been extensively studied, the movement of cells in the opposite direction has been less well documented. In particular, the question of whether T cells which have been activated by antigen in the periphery are more likely to return to the thymus had been raised but not clearly answered. We examined this issue by activating T cells present in the periphery with their cognate antigen, and assessing migration to the thymus. TCR-transgenic cells from OT-I mice (Thy1.2⁺), which recognise the ovalbumin peptide OVA_257–264 in the context of H-2K^b, were transferred into otherwise unmanipulated Thy1.1⁺ C57BL/6 mice. Recipient mice were injected i.v. with 5 μg peptide (SIINFEKL) approximately 24 hours later. The numbers of donor-derived (Thy1.2⁺) cells in the thymus and peripheral lymphoid tissue were determined. The results clearly show increased numbers of transgenic cells in the thymus 3 days after antigenic stimulation. However, since numbers of transgenic cells increased in the spleen and LN in about the same proportion, the data do not support the notion that there is highly increased selective migration of activated T cells to the thymus. Rather, they suggest that a sample of peripheral cells enters the thymus each day, and that the mature immigrants detected in the thymus merely reflect the contents of the peripheral T cell pool.     

Modulation of B and T cell subsets in mice treated with fractionated total lymphoid irradiation. III. Spleen and thymus dependency of B cell maturation processes

The role of spleen and thymus in maturation processes of B cell subpopulations was examined using mice which underwent fractionated total lymphoid irradiation (TLI). (BALB/BL)F₁ mice received 200 rds lymphoid irradiation daily for 8 days. Mice were spleen-shielded or -unshielded, or thymus-shielded or -unshielded during the irradiation. One day after termination of the irradiation, TLI-treated unshielded mice were reconstituted with 10 × 10⁶ spleen, thymus or bone marrow cells of normal untreated syngeneic mice. Two and a half months after termination of the treatment, the ability of the treated mice to produce in vivo anti-trinitrophenyl (TNP)-Ficoll antibodies, and the capability of spleen cells of those mice to respond in vitro to dextran sulfate and lipopolysaccharide was checked. In parallel, stained spleen cells were analyzed on the fluorescence-activated cell sorter. The results indicate that B cell maturation occurs only in mice where the spleens were shielded during TLI-treatment or in TLI-treated mice reconstituted with spleen cells of normal untreated mice. In these mice, the light scatter and the fluorescence distribution profiles were the same as those obtained from spleen cells of control mice: the treated mice gave a high anti-TNP-Ficoll antibody response and the proliferative response of the cells was low to dextran sulfate and high to lipopolysaccharide. Thymus shielding during TLI treatment or reconstitution of TLI-treated mice with thymus or bone marrow cells could not abolish the blockage of B cell maturation processes. These findings indicate that the spleen plays an obligatory role in B cell subset maturation pathways, whereas the thymus appears to play no essential role in these processes.     

Flexibility of the Thyroiditogenic T Cell Repertoire for Murine Autoimmune Thyroiditis in CD8-Deficient (B2m -/-) and T Cell Receptor VBC Congenic Mice

In murine experimental autoimmune thyroiditis (EAT), previous studies have revealed a highly adaptable thyroiditogenic T cell repertoire which involves both CD4⁺ and CD8⁺ T cells in the susceptible H2k strain. To further test this flexibility, congenic B10.K mice lacking CD8⁺ T cells (B2m -/-) or harboring 70% T cell receptor (TCR) V6 gene deletions (V6^C) were immunized with mouse thyroglobulin (MTg) and evaluated for EAT 28 days later. All 62m -/- mice developed moderate antibodies to MTg, and thyroidal inflammation was comparable to B10.K mice, averaging 35-40%. Spleen cells (SC) from MTg-immunized mice were then injected into syngeneic recipients after stimulation in vitro with MTg or with conserved, thyroxine (T4)- or thyronine (TO)- containing 12mer peptides, hT4(5), hT0(2553), or hT4(2553), derived from the primary hormonogenic sites at position 5 or 2553 of human Tg. As previously shown in another H2^k strain (CBA/J), all three peptides activated MTg-primed SC to transfer EAT in B10.K mice. hT4(5) and hT4(2553) were further tested in B10.K-V6^C and ß2m^- B10.K mice. Both peptides expanded thyroiditogenic T cells in either strain, resulting in severe thyroiditis in syngeneic recipients. That EAT can develop in the absence of CD8⁺ T cells or in the presence of a severely restricted TCR repertoire underscores the remarkable flexibility of the thyroiditogenic T cell profile in the susceptible k haplotype.     

Cell-mediated immune responses in vitro. V. A comparative study of in vitro immunogenicity of splenic lymphocytes, neoplastic lymphoid cells and fibroblats

This study compares the effectiveness of mouse lymphocytes, neoplastic lymphoid cells or fibroblasts in stimulating allogeneic cells to embark on an in vitro cytotoxic response, as measured by a ⁵¹Cr release assay. In addition, during ontogeny of mouse spleen cells, their capacity to stimulate in the mixed lymphocyte culture (MLR) was compared to their capacity to stimulate cytotoxic allograft responses. During ontogeny, there was amarked increase in the capacity of mouse spleen cells to stimulate mitotic responses in the MLR. In contrast, the magnitude of cytotoxic allograft responses induced by neonatal mouse spleen cells in the cytotoxic allograft system was comparable in magnitude to that induced by spleen cells of adult mice. The immunogenicity of subpopulations of mouse spleen cells was investigated. Mouse lymphoid cell populations, enriched for B or T lymphocytes or hemopoietic stem cells were equally immunogenic in vitro, as were myeloma or thymoma lymphoid cells. In contrast, mouse fibroblasts were found to elicit poor cytotoxic allograft responses. In fact, lymphoid cells were about 20–40-fold more immunogenic than fibroblasts.