Euthymic Recipients Research Articles

Auto-reconstitution of the T-cell compartment by radioresistant hematopoietic cells following lethal irradiation and bone marrow transplantation

In lethally irradiated bone marrow chimeras, part of the reconstituted T-cell compartment is derived from the irradiated host, but the detailed origin and functional activity of host-derived T cells has not been thoroughly analyzed. Herein, we determine the origin and function of radioresistant host-derived T cells. Lethally irradiated thymectomized or nonthymectomized C57BL/6 host mice were reconstituted with syngeneic bone marrow, itself incapable of generating T cells. Using fetal thymic organ cultures, bulk and limiting dilution assays on OP9-DL1 stromal cells, unambiguous cohorts of thymus-derived and peripheral T-cell-derived T cells were phenotypically characterized by flow cytometry and functionally characterized by their ability to participate in a T-cell-dependent antibody response. Both thymus-derived and peripheral T-cell-derived host T cells are functional and can reconstitute 35% of the normal T-cell pool. By comparing thymectomized vs nonthymectomized hosts, host-derived T cells were shown to comprise a major (70%) subpopulation of de novo generated, thymus-derived, polyclonal, naïve cells, and a minor subpopulation of surviving, peripheral, oligoclonal, memory-like cells. Unlike euthymic recipients, mice whose T cells were derived from surviving peripheral T cells were frequently incapable of mounting a T-cell-dependent antibody response. Host-derived thymocytes regenerated in an interleukin-7-dependent fashion from conventional DN2 thymocytes and their differentiation recapitulated normal thymic ontogeny. We characterized, for the first time, functional radioresistant DN2-phenotype thymic T-cell precursors, the T-cell progeny of which might provide a first line of defense against infections during the lymphopenic phase post-bone marrow transplantation.

374: Thymectomy does not abrogate long-term acceptance of MHC class I-disparate lung allografts

Background— We have previously reported that tolerance to class I disparate lung allografts in miniature swine could be induced using an intensive 12-day course of tacrolimus and that pretransplant sensitization with immunogenic MHC class I allopeptides failed to block the induction of tolerance. We also have previously reported the importance of the presence of the thymus in the induction of tolerance to isolated heart, kidney, and combined heart-kidney transplants. In this study, we examined the impact of thymectomy on tolerance induction. Methods— Orthotopic left lung transplantation was performed using MHC class I-disparate donors. The recipients received a 12-day course of high-dose tacrolimus (n = 6). Total thymectomies were performed in three of the swine, 21 days prior to transplantation. Lung grafts were monitored by chest radiography and serial open lung biopsy. Results— All euthymic recipients maintained their grafts over one year. None of the thymectomized recipients has shown graft loss in the six to ten months following transplantation. Although isolated lesions of obliterative bronchiolitis (OB) were occasionally seen in one thymectomized animal on biopsy, donor-specific unresponsiveness has been observed on assays of cell-mediated lymphocytotoxicity (CML) in all recipients. Moreover, co-culture CML assays have shown that recipient lymphocytes can strongly inhibit the normally robust response of naive recipient-matched lymphocytes to donor antigen. This inhibition was not seen when using stimulators primed with thirdparty antigens against appropriate targets. Conclusions— These data suggest that thymus-independent peripheral regulatory mechanisms may be sufficient to induce and maintain long-term acceptance of the lung allografts. 5 Address correspondence to: James S. Allan, MD, Massachusetts General Hospital, Blake 1570, 55 Fruit Street, Boston, MA 02114., Phone: 978-745-1581, Fax: 978-745-9024, E-mail: jallan@partners.org. 1This work was supported in part by grants from the Thoracic Surgery Foundation for Research and Education (Sahara/Allan), the International Society for Heart and Lung Transplantation (Shoji), the American Surgical Association Foundation (Allan), and the National Institutes of Health 1RO1-HL67110-01-A1 (Allan). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errorsmaybe discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript Transplant Proc. Author manuscript; available in PMC 2007 July 15. Published in final edited form as: Transplant Proc. 2006 December ; 38(10): 3253–3255. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript

Thymectomy Does Not Abrogate Long-Term Acceptance of MHC Class I-Disparate Lung Allografts in Miniature Swine

We have previously reported that tolerance to class I disparate lung allografts in miniature swine could be induced using an intensive 12-day course of tacrolimus and that pretransplant sensitization with immunogenic MHC class I allopeptides failed to block the induction of tolerance. We also have previously reported the importance of the presence of the thymus in the induction of tolerance to isolated heart, kidney, and combined heart-kidney transplants. In this study, we examined the impact of thymectomy on tolerance induction in lung transplantation. Methods Orthotopic left lung transplantation was performed using MHC class I-disparate donors. The recipients received a 12-day course of high-dose tacrolimus ( n = 6). Total thymectomies were performed in three of the swine 21 days prior to transplantation. Lung grafts were monitored by chest radiography and serial open lung biopsy. Results All euthymic recipients maintained their grafts for over 1 year. None of the thymectomized recipients has experienced graft loss in the 6 to 10 months following transplantation. Although isolated lesions of obliterative bronchiolitis were occasionally seen in one thymectomized animal on biopsy, donor-specific unresponsiveness has been observed on assays of cell-mediated lymphocytotoxicity in all recipients. Moreover, co-culture assays have shown that recipient lymphocytes can strongly inhibit the normally robust response of naïve recipient-matched lymphocytes to donor antigen. This inhibition was not seen when using stimulators primed with third-party antigens against appropriate targets. Conclusions These data suggest that thymus-independent peripheral regulatory mechanisms may be sufficient to induce and maintain long-term acceptance of the lung allografts.

CD4+CD25+ Regulatory T Cells Enhance Immune Reconstitution Following Allogeneic Hematopoietic Cell Transplantation by Protecting Thymic and Lymphoid Compartments from Graft-Versus-Host Disease Damage without Impacting T Cell Repertoire Development.

Regulatory T cells (Treg) protect from acute graft-versus-host disease (GvHD) in murine models of major-MHC mismatched hematopoietic cell transplantation (HCT) presumably by dampening the proliferation of mature effector T cells. It is unclear whether the effect of Treg on effector T cells is a selective or nonselective process or if Treg regulate the process of intrathymic and peripheral T cell maturation and selection following HCT, particularly given the intrinsic link of GvHD and immune reconstitution. We previously showed that Treg improved the quantitative and functional lymphoid reconstitution in a murine model of HCT. In the current study, we hypothesize that Treg prevent thymic and lymphoid damage from GvHD, leading to enhanced lymphoid reconstitution. Lethally-irradiated adult thymectomized Balb/c (H2d) recipients were transplanted with wild-type FVB (H2q) T cell depleted bone marrow (TCD-BM) cells and CD4+/CD8+ cells (Tcon), the latter to induce GvHD, with or without donor Treg given at a 1:1 dose ratio with Tcon. At day 30, when all groups had reached full donor chimerism, transplant recipients were challenged with murine CMV (5×105 pfu/mouse) intraperitoneally. At day 90, survival for thymectomized groups with TCD-BM alone, with Tcon, or with Tcon+Treg was 78%, 0%, and 45%, respectively, compared to 100%, 0%, and 86% survival in their respective euthymic infected counterparts (p<0.05 for thymectomized vs euthymic Treg groups). Elispot for Interferon-γ showed CMV-specific donor responses in all infected groups. CMV viral titers in the liver and kidney 2 weeks after infection was lower in recipients that received Treg compared to animals that received Tcon alone. Compared to euthymic transplant controls, thymectomized animals had higher viral titers in the liver, lungs, and kidneys in all groups. Uninfected thymectomized mice in the respective groups served as controls to separate the effect of CMV infection and GvHD on survival. All animals, infected or uninfected, that received Treg had no evidence of clinical GvHD while animals that received Tcon alone had significant GvHD. In euthymic recipients, gross and histologic examination confirmed the general preservation of thymic integrity and architecture in animals that received Treg compared with smaller involuted thymuses partially replaced by adipose tissue in animals that received Tcon alone. T cell repertoire assessed by V-beta TCR screening with FACS analysis showed a polyclonal distribution in animals with or without Treg. Spectratyping at day 30 post-transplantation showed that Treg had no significant impact on the TCR repertoire diversity in animals which received Tcon. Based on survival of a subset of infected thymectomized animals that received Treg, we evaluated the impact of Treg on secondary lymphoid organs following HCT. Animals without transferred Treg had significant splenic and lymph node fibrosis and hypoplasia with a reduction in T cell numbers due to GvHD. Our findings indicate that Treg indirectly enhance immune reconstitution by protecting the thymic and secondary lymphoid compartments from GvHD damage, allowing the generation and peripheral expansion of lymphoid cells without impacting the diversity of T cell repertoire.

Role of Thymus in Operational Tolerance Induction in Limb Allograft Transplant Model

In this study, we evaluated the role of host thymus in tolerance induction in composite tissue allografts (CTA) across major histocompatibility complex (MHC) barrier during a 7-day alphabeta- T-cell receptor (TCR)/ cyclosporine A (CsA) protocol. A total of 62 limb allograft transplants were studied. Euthymic (group A) and thymectomized (group B) Lewis recipients (LEW, RT1(1)) received vascularized hind-limb allografts from hybrid Lewis x Brown-Norway (F1), (LBN, RT1(1+n)) donors. Mixed lymphocyte reaction (MLR) and skin grafting assessed donor-specific tolerance in vitro and in vivo, respectively. Flow cytometry determined the efficacy of immunosuppressive protocols and the presence of donor-specific chimerism. Immunocytochemistry revealed the presence of donor-specific cells in the lymphoid organs of recipients. Isograft transplants survived indefinitely. For thymectomized rats, the median survival time (MST) of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR extended MST to 16 days, and CsA therapy extended it to 30 days. Using the alphabeta-TCR/CsA protocol, the MST of allografts was 51 days. For euthymic rats, the MST of limb allograft in non-treated recipients was 7 days; monotherapy with alphabeta-TCR or CsA extended MST to 13 or 22 days, respectively. Treatment with alphabeta-TCR/CsA resulted in indefinite allografts survival (MST=370 days). MLR and skin grafting confirmed donor-specific tolerance in euthymic recipients. Flow cytometry showed stable chimerism in the euthymic rats and transient chimerism in thymectomized limb recipients. Immunoperoxidase staining revealed the persistence of donor-derived cells in the lymphoid tissues of euthymic recipients. We found that the presence of thymus was imperative for the induction of donor-specific tolerance in rat hind-limb composite tissue allografts using a alphabeta-TCR/CsA protocol.

Role of the Thymus in Transplantation Tolerance in Miniature Swine: V. Deficiency of the Graft-to-Thymus Pathway of Tolerance Induction in Recipients of Cardiac Transplants

We have previously shown that both thymic immigrants (graft to thymus pathway) and thymic emigrants (thymus to graft pathway) are involved in tolerance to renal allografts in miniature swine treated with a short course of calcineurin inhibitors. This study investigates the role of these pathways in cardiac transplant survival in recipients treated with a short course of tacrolimus. Eleven animals received two-haplotype fully MHC-mismatched cardiac grafts with a 12-day course of tacrolimus. Recipients were thymectomized on day -21 (n=5) or day 0 (n=3), or were left euthymic (n=3). Two of the day -21 thymectomized animals received a day 0 host-MHC matched thymocyte infusion. Euthymic recipients of cardiac grafts treated with an immunosuppressive regimen identical to that previously shown to induce tolerance in euthymic recipients of renal allografts all rejected their grafts. Although no animal became tolerant, animals that were euthymic or thymectomized on day 0, as well as recipients of day 0 host-type thymocyte infusions following thymectomy on day -21, developed donor-specific hyporesponsiveness and maintained their cardiac grafts for markedly prolonged periods. In contrast, all animals thymectomized on day -21 that did not receive thymocyte infusions developed strong antidonor CTL responses and rejected their grafts by day 35. The graft-to-thymus pathway that plays an important role in tolerance induction to renal allografts appears to be relatively deficient in recipients of cardiac grafts. Strategies to increase donor antigen migration to the host thymus might therefore assist in tolerance induction to cardiac allografts.

Chronic allograft nephropathy in athymic nude rats after adoptive transfer of primed T lymphocytes

The impact of presensitized T lymphocytes on the development of chronic allograft nephropathy (CAN) was investigated in nude athymic LEW.RNU recipients of F344 renal allografts. The recipients (n = 8) were reconstituted with 5 x 10(7) T lymphocytes primed against donor skin grafts to induce graft rejection. LEW.RNU (n = 8) and euthymic LEW recipients (n = 6) which underwent no further intervention after transplantation served as control groups. Adoptive transfer of primed T cells induced CAN in LEW.RNU rats. Their kidney function decreased progressively. After 90 days a moderate glomerulopathy, tubular atrophy and interstitial fibrosis were observed, vascular changes were only mild or absent. Cellular infiltrates were predominated by CD4+ T cells and ED1+ macrophages. Deposition of tenascin and laminin was enhanced. Grafts of euthymic recipients displayed only mild signs of CAN according to the Banff criteria. These data implicate an important role for the cellular immune response in the development of CAN.

Vascularized thymic lobe transplantation in miniature swine: thymopoiesis and tolerance induction across fully MHC-mismatched barriers.

As the major site of self-nonself discrimination in the immune system, the thymus, if successfully transplanted, could potentially carry with it the induction of central tolerance to any other organ or tissue from the same donor. We have recently developed a technique for transplantation of an intact, vascularized thymic lobe (VTL) in miniature swine. In the present study, we have examined the ability of such VTL allografts to support thymopoiesis and induce transplantation tolerance across fully MHC-mismatched barriers. Six miniature swine recipients received fully MHC-mismatched VTL grafts with a 12-day course of tacrolimus. Three of these recipients were thymectomized before transplantation and accepted their VTL allografts long-term, with evidence of normal thymopoiesis. In contrast, three euthymic recipients rejected their VTL allografts. Donor renal allografts, matched to the donor VTL grafts, were transplanted without immunosuppression into two of the three thymectomized recipients, and one of the three euthymic recipients. These renal allografts were accepted by thymectomized recipients, but rejected by the euthymic recipient in an accelerated fashion. This study thus demonstrates that successful transplantation of a vascularized thymus across a fully MHC-mismatched barrier induces tolerance in this preclinical, large-animal model. This procedure should enable studies on the role of the thymus in transplantation immunology as well as offer a potential strategy for tolerance induction in clinical transplantation.

Composite ‘Thymoheart’ Transplantation Improves Cardiac Allograft Survival

We have generated a novel composite organ, the thymoheart, which facilitates the contemporaneous transfer of fully vascularized and functional donor thymic tissue to the host at the time of cardiac transplantation. Composite thymoheart allografts were prepared in MHC-inbred miniature swine by implanting autologous thymic tissue into donor hearts 60–90 days before organ procurement. Thymoheart allografts and unmanipulated control hearts were then transplanted into three groups, each treated with the same 12 days of cyclosporine. MHC-matched thymohearts transplanted into euthymic recipients had a minimum survival ranging between 72 and 194 days vs. 42–64 days for unmanipulated control hearts (p = 0.02). MHC class I-disparate thymohearts transplanted into euthymic recipients had a minimum survival ranging between 64 and 191 days vs. 30–55 days for unmanipulated control hearts (p = 0.01). MHC class I-disparate thymohearts transplanted into thymectomized recipients survived between 41 and 70 days vs. 8–27 days for unmanipulated control hearts (p = 0.01). Cellular and humoral functional assays, and skin grafting, confirmed the presence of donor-specific hyporesponsiveness in long-term thymoheart allografts recipients. The transfer of vascularized, functional donor thymic tissue to the host at the time of cardiac transplantation may provide a novel approach to the induction of tolerance in human heart transplant recipients.

Piscine islet xenotransplantation.

Tilapia, a teleost fish species with large anatomically discrete islet organs (Brockmann bodies; BBs) that can be easily harvested without expensive and fickle islet isolation procedures, make an excellent donor species for experimental islet xenotransplantation research. When transplanted into streptozotocin-diabetic nude or severe combined immunodeficient mice, BBs provide long-term normoglycemia and mammalian-like glucose tolerance profiles. However, when transplanted into euthymic recipients, the mechanism of islet xenograft rejection appears very similar to that of islets from "large animal" donor species such as the very popular fetal/neonatal porcine islet cell clusters (ICCs). Tilapia islets are more versatile than ICCs and can be transplanted (1) into the renal subcapsular space, the cryptorchid or noncryptorchid testis, or intraportally as neovascularized cell transplants; (2) as directly vascularized organ transplants; or (3) intraperitoneally after microencapsulation. Unlike the popular porcine ICCs, BBs function immediately after transplantation; thus, their rejection can be assessed on the basis of loss of function as well as other parameters. We have also shown that transplantation of tilapia BBs into nude mice can be used to study the possible implications of cross-species physiological incompatibilities in xenotransplantation. Unfortunately, tilapia BBs might be unsuitable for clinical islet xenotransplantation because tilapia insulin differs from human insulin by 17 amino acids and, thus, would be immunogenic and less biologically active in humans. Therefore, we have produced transgenic tilapia that express a "humanized" tilapia insulin gene. Future improvements on these transgenic fish may allow tilapia to play an important role in clinical islet xenotransplantation.

Control of T cell hyperactivation in IL-2-deficient mice by CD4(+)CD25(-) and CD4(+)CD25(+) T cells: evidence for two distinct regulatory mechanisms.

In IL-2-deficient mice, antigen-activated CD4 T cells accumulate and cause lethal immune pathology. Wild-type cells of hematopoietic origin present in the same animal are able to prevent this hyperactivation of T cells, but the mechanisms and cells controlling the IL-2-deficient cells are unknown. Here we show that IL-2(-) CD4 cells with an ovalbumin-specific transgenic TCR (IL-2(-) OVAtg) undergo both clonal expansion and clonal contraction when transferred to euthymic recipients and challenged with antigen, but continuously expand in athymic hosts. Cotransfer of wild-type CD4 T cells prevents the accumulation of IL-2-deficient cells. On the residual IL-2(-) TCRtg cells CD69 and CD25 are up-regulated, suggesting that activation per se is not suppressed and that the cells had received an IL-2 signal. Since IL-2 is able to restore the defective antigen-induced cell death (AICD) of IL-2-deficient T cells in vitro, paracrine IL-2 provided by the wild-type CD4 cells may thus be able to allow clonal contraction of IL-2-deficient cells also in vivo. Interestingly however, regulatory CD4(+)CD25(+) cells also efficiently contain the clone size of antigen-stimulated IL-2-deficient T cells. Since CD4(+)CD25(+) cells do not produce IL-2, this suggests a mechanism of suppression distinct from paracrine IL-2 delivery. In keeping with this, the residual IL-2(-) TCRtg cells recovered after cotransfer of regulatory CD4(+)CD25(+) cells do not show increased CD25 or CD69 expression, suggesting that they had not received paracrine IL-2 and that clonal containment occurred at the level of initial activation rather than clonal contraction by AICD. IL-2 deficiency therefore may upset T cell homeostasis by two distinct mechanisms: the failure to program expanding T cells for apoptosis, and the failure to generate functional CD4(+)CD25(+) regulatory cells.

Cell Therapy for Diabetes Using Piscine Islet Tissue

The islet tissue, called Brockmann bodies, in certain teleost fish is anatomically distinct from the pancreatic exocrine tissue and can be easily identified macroscopically. Expensive islet isolation procedures, such as required when procuring islet tissue from mammalian pancreases, are unnecessary. Tilapia islets transplanted into diabetic athymic nude mice will produce long-term normoglycemia and mammalian-like glucose tolerance profiles. Encapsulated tilapia islets function well after transplantation into euthymic recipients. Additionally, tilapia are potentially ideal xenogeneic donors because of markedly lower donor production costs, minimal islet procurement costs, and possibly decreased xenozoonotic potential relative to mammalian donors. Tilapia islets appear to be appropriately glucose responsive with high insulin output, can be cryopreserved, and are much more resistant to hypoxia than mammalian islets. Because tilapia and human insulin differ by 17 amino acids, we have cloned, sequenced, and modified the tilapia insulin gene by site-directed mutagenesis resulting in a tilapia insulin gene that codes for "humanized" insulin while still maintaining all of the tilapia regulatory (noncoding) sequences. We have now produced small numbers of founder transgenic tilapia with incorporation of our humanized tilapia insulin gene construct, and we have shown transmission and expression of the transgene in the beta cells and serum of their Fl offspring. Our ultimate goal is to achieve homologous recombination and to breed for homozygosity for the hybrid insulin gene. Subsequent generations of transgenic tilapia will undergo a program of genomic optimization selecting for growth, survival, and fecundity as well as stability of the transgene. Islets from the resulting transgenic fish, after extensive characterization, could be harvested, encapsulated, and then transplanted into diabetic patients.

Mechanisms of tolerance induction and prevention of cardiac allograft vasculopathy in miniature swine: The effect of augmentation of donor antigen load

Objective: Cotransplantation of a donor kidney along with a heart allograft can induce tolerance to both organs and prevent cardiac allograft vasculopathy in miniature swine. To determine whether the tolerogenic effect of donor kidney cotransplantation was due to an effect specific to the kidney graft or to an increase in donor antigen load, we compared heart-kidney recipients with recipients receiving two class I disparate hearts or with recipients receiving donor peripheral mononuclear cells at the time of isolated heart transplantation. Methods: Recipients of major histocompatibility complex class I disparate allografts received 12 days of cyclosporine (INN: ciclosporin; 10-13 mg/kg administered intravenously on days 0-11). Group 1 animals received a heart alone (n = 5). Group 2 animals received heart and kidney allografts (n = 4). Group 3 animals received two major histocompatibility complex–matched heart allografts (n = 4). Two double-heart recipients were thymectomized 21 days before transplantation. Group 4 animals received a heart allograft and an infusion of high-dose donor peripheral blood leukocytes (2.5 × 10 9 cells/kg, n = 2). Results: Vasculopathy developed in group 1 recipients and the allografts were rejected within 55 days. Group 2 recipients accepted their heart and kidney allografts indefinitely without vasculopathy. Euthymic recipients from group 3 accepted their hearts long-term (>190 and >197 days), but vascular lesions developed. In thymectomized recipients from group 3, the hearts were rejected in 63 and 96 days with severe vasculopathy. Group 4 recipients demonstrated transient macrochimerism but their hearts were rejected within 47 and 63 days. Conclusions: The beneficial effects of donor kidney cotransplantation on cardiac allograft survival and prevention of cardiac allograft vasculopathy are likely to involve both an increase in donor antigen load and an effect specific to the kidney allograft. (J Thorac Cardiovasc Surg 2000;119:709-19)

Experimental orthotopic corneal xenotransplantation in the rat. Mechanisms of graft rejection.

Orthotopic penetrating guinea pig to rat and chicken to rat corneal xenografts were performed to examine the nature of the host response. Guinea pig to rat xenografts failed at a median of day 3 after surgery. A similar but slightly accelerated pattern of failure was seen in guinea pig xenografts performed in prevascularized recipient rat corneas. Chicken to rat xenografts failed at a median of day 2 after grafting. Rat corneal isograft controls survived indefinitely. Corneal endothelial cells were visible by silver staining on the xenografts immediately after operation, which indicates that failure was not due to loss of these cells during surgery. Histopathology and immunoperoxidase staining indicated that xenograft failure in euthymic recipients was characterized by early corneal epithelial and endothelial cell damage, granulocytic infiltration, and hemorrhage from recipient corneal and iris capillaries, followed at 7-14 days by infiltration with T cells, macrophages, and eosinophils. An accelerated pattern of graft failure was also observed in guinea pig grafts into homozygous nude rat recipients, which suggests that preformed anti-donor antibody and complement were responsible for some of the early graft damage. Flow cytometry demonstrated the presence of pre-existing natural antibodies to guinea pig and chicken lymphocytes and erythrocytes, as expected from other studies. Immunohistochemistry showed the presence of rat IgG2a, IgG1, and IgM, but not IgD deposited on grafts in immunocompetent recipients on the first postoperative day. We conclude that orthotopic corneal xenografts undergo substantial accelerated damage mediated by pre-existing antibody, followed at 7-14 days by a cell-mediated response that causes further destruction.

Membrane CD45R isoform exchange on CD4 T cells is rapid, frequent and dynamic in vivo.

CD4 T cells bearing high (240-190 kDa) and low (180 kDa) molecular mass isoforms of the leukocyte common antigen CD45 define functionally distinct subsets which have been equated with naive and memory T cells. In the rat, CD4 T cells expressing a high molecular mass isoform [identified by monoclonal antibody MRC-OX22 (anti-CD45RC)] exchange this for the 180 kDa molecule (CD45RC-) when stimulated by antigen. Here we show, by transferring mature allotype-marked CD45RC- CD4 T cells (depleted of immature Thy-1+ CD45RC- recent thymic emigrants) into normal euthymic recipients, that many T cells re-express the high molecular mass isoform in less than 6 h. By 24 h, 30-60% of CD45RC- CD4 T cells became CD45RC+; within a week the entire cohort appeared to exchange the low for the high molecular mass isoform. Isoform exchange was dynamic and many CD4 T cells returned once again to the CD45RC- state. CD45RC- CD4 T cells declined in number more rapidly than the CD45RC+ subset after transfer. The results suggest that CD45R isoforms distinguish between resting T cells (CD45RC+) and those which have encountered antigen in the recent past. CD45R isoforms would appear to be unsuitable markers of naive and memory T cells.

CD4+ T cell-mediated rejection of major histocompatibility complex class I-disparate grafts: a role for alloantibody.

Experimental studies of the T cell requirement for rejection of class I major histocompatibility complex (MHC)-disparate grafts have generated controversy over both the autonomy of CD8+ T cells and the mechanism whereby CD4+ T cells are able to independently mediate rejection. In this study of rejection of RT1Aa class I MHC-disparate rat cardiac and skin allografts by high-responder PVG RT1u recipients, we show that elimination of CD8+ T cells [by anti-CD8 monoclonal antibody (mAb) administration in vivo] fails to prolong graft survival, whereas partial depletion of CD4+ T cells (by anti-CD4 mAb treatment) markedly delays rejection of class I-disparate heart grafts, and marginally prolongs survival of skin grafts. Anti-CD4-treated PVG-RT1u athymic nude rats reconstituted with CD8+ T cells failed to reject class I-disparate skin grafts for several weeks and eventual rejection correlated with re-emergence of a small number of donor derived CD4+ T cells. Conversely, anti-CD8-treated nude rats reconstituted with CD4+ T cells alone rapidly rejected class I-disparate skin grafts. Passive transfer of anti-class I immune serum to anti-CD4-treated euthymic recipients promptly restored their ability to specifically reject a class I-disparate heart graft. Similarly, passive transfer of immune serum to PVG-RT1u nude rats bearing skin allografts caused destruction of class I-disparate but not third-party grafts. These results demonstrate that CD4+ T cells are both necessary and sufficient to cause rejection of class I-disparate heart and skin grafts in this model and that CD4+ T cell-dependent alloantibody plays a decisive role in effecting rejection.

The Neuro-endocrine Component of the Rat Thymus: Studies on Cultured Thymic Fragments Before and After Transplantation in Congenitally Athymic and Euthymic Rats

An immunohistochemical study was done for the presence of tyrosin hydroxylase (noradrenergic innervation), neuron-specific protein PGP9.5, and anterior pituitary hormones (β-subunit of follicle-stimulating hormone, growth hormone, β-subunit of luteinizing hormone, prolactin, and β-subunit of thyroid-stimulating hormone) in cultured thymic fragments before and after transplantation in congenitally athymic and euthymic rats. The cultured thymic fragments consisted of epithelial cells and were depleted of lymphocytes. After implantation in syngeneic and allogeneic athymic recipients and in syngeneic euthymic recipients, a recovery of the original architecture was found within 6 weeks; rejection occurred within 3 weeks for allogeneic transplantation in euthymic rats. During culture nerve-like profiles almost disappeared from the tissue, and reappeared simultaneously with the influx of host-derived cells and the restoration of the original thymic architecture. A high immunoreactivity for hormones and PGP9.5 was found in epithelial cells after culture and in the first phase after transplantation. These epithelial cells may represent precursor-epithelial cells, based on their unusual ultra-structure and combined expression of markers that in the normal thymus occur only on subcapsular/medullary epithelium or on cortex epithelium. These data indicate a potential role of the neuroendocrine function of the thymus during restoration of the thymus architecture starting from precursor-like epithelial cells.

Functional maturation of recent thymic emigrants in the periphery: development of alloreactivity correlates with the cyclic expression of CD45RC isoforms.

The transition from fully developed CD4+CD8- single-positive (SP) thymocytes into fully mature recirculating peripheral T cells is both poorly understood with regard to the expression of restricted isoforms (CD45R) of the leukocyte common antigen and in terms of T cell function. The present investigation monitored the extrathymic development of CD4+CD8- SP thymocytes in euthymic recipients using allotype-marked donor cells and monoclonal antibody OX22 which recognizes an epitope on the C exon of rat CD45R. We established that donor-derived cells in the blood 1 day later bore the phenotype of the injected SP thymocytes (CD4+ Thy-1+ CD45RC-). T cells with the identical phenotype were also present in the thoracic duct lymph of uninjected rats, suggesting that the Thy-1+ CD45RC- T cells represent recent thymic emigrants (RTE) which have migrated to the periphery of their own accord. During extrathymic maturation donor-derived peripheral RTE lost Thy-1 within 3 days and expressed the CD45RC+ high molecular weight isoform by day 7; between days 8 and 14 a proportion (25%-30%) of the donor cells once again lost the high molecular weight isoform (CD45RC-). The transition of SP (CD45RC-) thymocytes to fully mature CD45RC+ CD4 T cells via intermediate peripheral RTE was accompanied at each stage by an increased ability of the maturing T cells to induce skin allograft rejection. Unexpectedly, the subsequent loss of the high molecular weight isoform, following presumed antigen encounter, was associated with a significant reduction in the ability of this Thy-1-CD45RC- subpopulation to effect graft rejection. The cyclic expression of CD45RC isoforms on both immature and mature CD4 T cells and the fact that the low molecular weight isoform was found in the periphery on both RTE (unquestionably naive) and antigen-experienced CD4 T cells, makes it unlikely that this isoform uniquely identifies memory T cells, at least in the rat.

Transplantation of cultured thymic fragments in congenitally athymic and euthymic rats. Culture with deoxyguanosine or cyclosporin A does not influence the histologic characteristics and outcome after transplantation in syngeneic and allogeneic combinations.

Cultured thymic fragments (CTF) from WAG/CPB (RT1u) and DA/01a (RT1a) rats were prepared in the presence or absence of 2'deoxyguanosine or cyclosporin A, and subsequently transplanted under the kidney capsule of congenitally athymic and euthymic WAG/CPB recipients. The rationale of the culture supplements was that these may affect the disappearance of medullary dendritic cells, with subsequent induction of allotolerance. However, the immunohistology of the CTF showed more RT1 class II-positive cells than keratin-positive cells, indicative of the maintenance of dendritic cells. Grafts in athymic animals showed the recovery of the original thymic architecture within 6 weeks after transplantation. The influx of host-derived lymphocytes was accompanied by an influx of dendritic cells in the medulla-like area and macrophages in the cortex. A similar recovery was observed for syngeneic CTF in euthymic recipients. In addition lymphocytic infiltration was seen in the connective tissue surrounding the epithelial areas. Allogeneic grafts in euthymic animals were rejected within 3 weeks after transplantation. This outcome of the transplanted CTF under different conditions was not affected by the supplementation of the thymic culture before transplantation with 2'deoxyguanosine or cyclosporin A. We conclude that there is no tolerance induction after transplantation in euthymic allogeneic rats of CTF prepared in the presence of 2'deoxyguanosine. This conclusion is in contrast to data in the mouse, which may be explained by the maintenance of dendritic cells during culture. A chimaeric state of donor-derived epithelium and host-derived dendritic cells is obtained by transplantation of allografts in athymic rats.

Experimental Salmonella typhimurium infections in rats

The protective effect of primed lymphocytes against a lethal dose of Salmonella typhimurium was studied in athymic and euthymic LEW rats. Primed lymphocytes were obtained by inoculating euthymic and thymus‐grafted animals with a non‐lethal dose of Salmonella typhimurium. Four weeks after the infection, spleen lymphocytes were separated by panning technique and antibody‐coated magnetic microsphere separation using antibodies to pan T and pan B lymphocytes and subsequent sorting in a fluorescense activated cell sorter by means of monoclonal antibodies against CD4+ and CD8+ cells. Euthymic and athymic rats were injected with different doses of primed pan B, pan T, CD4+ and CD8+ T lymphocytes before inoculation with a lethal bacterial dose. Most of the animals treated with pan B, pan T or CD8+ cells died within two weeks. After treatment with primed CD4+ cells, only six of 39 animals died. Doses as low as 104 cells from both euthymic or thymus‐grafted animals were effective, and athymic and euthymic recipients survived equally well. Four weeks after the infection both athymic and euthymic animals housed very few bacteria and had high antibacterial antibody titres. The percentages of splenic and lymph node CD4+ cells in the athymic rats were comparable to those found in the euthymic animals. The study shows that primed CD4+ lymphocytes even in very low doses are able to induce immunity against a Salmonella typhimurium infection.