Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell-mediated rejection response declines with advanced age

The present investigation explored age-related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H-2^b and H-2^bxH-2^k mice were engrafted with major histocompatibility complex (MHC) class II-disparate bm12 skin, rejection of which requires CD4⁺ T cells, and MHC class I-disparate bm1 skin, rejection of which requires CD8⁺ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Ia^bm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of K^bm1 CTL from T cells of aged mice. The alterations in Ia^bm12 allospecific responses were not attributable to quantitative changes in CD4⁺ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Ia^bm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4⁺ T cell-mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.     

CD4 T cells can reject major histocompatibility complex class I-incompatible skin grafts

We have re-investigated the roles of CD4 and CD8 T cell subsets in skin graft rejection across a single class I MHC disparity. Recipient mice were transplanted with skin from donors transgenic for the class I MHC molecule K^b. As expected, CD8 T cells were sufficient for rapid injection; but surprisingly, CD4 T cells were also competent to do the same. Rejection was dependent on one or the other subset, since elimination of both resulted in indefinite graft survival. The possibility that alloantibody was the downstream effector of CD4 mediated rejection was excluded because CD8-depleted mice rendered B cell deficient still rejected rapidly, but T cell-depleted recipients with pre-existing high titers of alloantibody were unable to do so. In addition, if CD4 cells act to reject by recruiting and/or activating macrophages then this was not dependent on CR3, IFN-γ or TNF-α. Transplantation of skin grafts where the MHC class I disparity was at the level of passenger leukocytes only, demonstrated that transient bystander damage could occur, but that this was insufficient to result in full rejection. We surmise that for CD4 T cells to reject an MHC class I-incompatible graft it is necessary that an appropriate allogeneic peptide is processed and presented in the context of recipient MHC class II. CD4 T cells from B6 mice may fail to reject skin from MHC class I mutants because of the lack of such MHC class II-restricted presentation.     

Impact of donor MHC class I or class II antigen deficiency on first- and second-set rejection of mouse heart or liver allografts

The influence of donor major histocompatibility complex (MHC) class I- or class II-deficiency on the initiation of first- and second-set rejection of mouse heart and liver allografts was examined. C3H (H-2^k) mice received heterotopic cardiac or orthotopic liver grafts from unmodified B10 (H-2^b), B6 (H-2^b), b2m (H-2^b; class I deficient) or AB⁰ (H-2^b; class II deficient) donors. Organ survival was also investigated in C3H recipients that had been presensitized by a normal B10 skin graft 2–3 weeks before heart or liver transplantation. The absence of cell surface MHC class I or class II resulted in significant prolongation of primary cardiac allograft survival. Three of seven (43%) MHC class I-deficient, and two of five (40%) class II-deficient heart grafts were accepted indefinitely (survival time >100 days). Thus both MHC class I and class II molecules appear to be important for the elicitation of first-set rejection in the heart allograft model. All liver allografts survived >100 days in normal recipients. In C3H recipients that had been presensitized by a B10 skin graft, however, both heart and liver grafts from AB⁰ (class II deficient) donors underwent accelerated rejection (median survival time [MST] 3 and 4 days, respectively). In contrast, liver grafts from class I-deficient mice (b2m) were still accepted indefinitely by B10 skin-presensitized C3H recipients, whereas class I-deficient hearts survived significantly longer than those from class II-deficient or normal donors. These data demonstrate that the expression of donor MHC class I, and not class II is crucial in initiating second-set organ allograft rejection. In vitro monitoring revealed that at the time of organ transplant, both splenocytes and serum of the skin-presensitized animals displayed high cytotoxicity against AB⁰ (class II-deficient) but not against b2m (class I-deficient) targets.     

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2^b or H-2^d Tgβ-gal mice) expressed the mHAg like β-galactosidase (β-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tgβ-gal skin by a β-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous β-gal shed by keratinocytes. Indeed, our results showed that 1) H-2^b Tgβ-gal skin was rejected by H-2^bxs and H-2^bxd recipients; 2) rejection was mediated by β-gal-specific CD8⁺ CTL effectors; and 3) H-2^bxd mice having rejected H-2^b Tgβ-gal skin generated β-gal-specific CTL restricted by H-2^b and H-2^d class I molecules and rejected subsequently grafted H-2^d Tgβ-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous β-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.     

Graft rejection by T cells not restricted by conventional major histocompatibility complex molecules

The appropriate crosses of mice lacking conventional major histocompatibility complex (MHC) class I or class II molecules generate single- and double-deficient offspring. These were used as donors for skin grafts across major plus minor, or just minor, histocompatibility differences. Surprisingly, in the two circumstances, there was a rapid rejection of grafts lacking both MHC class I and class II molecules. Rejection was mediated by thymically derived CD4⁺ T cells of the host. We provide evidence that these T cells recognize an unconventional ligand, capable of activating a pre-formed T cell compartment but incapable of positively selecting it. The existence of this unexpected rejection phenomenon should serve to caution those aiming to engineer “universal donor” cells by simply abrogating expression of MHC class I and class II molecules.     

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 RT1A^a class I MHC-disparate rat cardiac and skin allografts by high-responder PVG RT1^u 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-RT1^u 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-RT1^u 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.     

TAP1-/- mice present oligoclonal BV-BJ expansions following the rejection of grafts bearing self antigens

Our previous work showed that transporter associated with antigen processing 1 (TAP1)^–/– (H‐2^b) mice rejected grafts from H‐2^b mice which display a normal density of class I major histocompatibility complex (MHC) molecules at the cell surface. Our results indicated that H‐2^b molecules themselves may be a target in this kind of rejection and that CD4⁺ T cells play a major role in this autoreactive process. Our data also suggested that TAP1^–/– mice, in addition to the well‐recognized phenotype of class I and CD8⁺ T‐cell deficiency, present a functional alteration in their autoreactive CD4⁺ T‐cell repertoires. In this model of inflammatory autoreactivity to modified self, we have analysed T‐cell receptor (TCR) V‐beta–J‐beta (BV‐BJ) usage by complementarity determining region 3 (CDR3) length spectratyping in splenocytes from naïve TAP1^–/– mice and transplanted TAP1^–/– mice that rejected B6 heart grafts or responded to synthetic self H‐2K^b peptides. Importantly, oligoclonal T‐cell expansions shared by different animals were detected in the peripheral T‐cell repertoire of transplanted TAP1^–/– mice. Such public expansions were also induced in vitro by H‐2K^b peptides, suggesting that dominant class I peptides can induce preferential expansions of restricted T‐cell populations during rejection. Some of these public T‐cell expansions were also detected in transplanted mice even before in vitro stimulation with peptides, indicating that post‐transplantation expansion of these populations had occurred in vivo. The functional activity of these T‐cell populations awaits elucidation, as do the underlying mechanisms involved in the inflammatory autoreactive process, in TAP1^–/– mice.     

Antigen-Specific Killer Polylactic-Co-Glycolic Acid (PLGA) Microspheres Can Prolong Alloskin Graft Survival in a Murine Model

Background: The strategy of specifically depleting antigen-specific T cells can potentially be used for the treatment of allograft rejection and autoimmunity because it does not suppress the overall immune systems.

Methods: In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling major histocompatibility complex (MHC) class I antigens and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto PLGA microspheres. A modified double-emulsion method was used for the preparation of cell-sized PLGA microspheres. H-2K^b/peptide monomers were generated in-house and analyzed through flow cytometry. The killer PLGA microspheres were administered intravenously into BALB/c mice (H-2K^d) that had previously been grafted with skin squares from C57BL/6 mice (H-2K^b). Tumor cell challenge and third-party mixed lymphocyte culture were used to assess the general immune functions of host.

Results: The alloskin graft survival was prolonged by 4?days. The killer PLGA microspheres could specifically deplete the H-2K^b alloantigen-reactive CD8⁺ T cells that infiltrated into the alloskin graft but not CD4⁺ T cells, without impairment of host overall immune function.

Conclusions: Here, we initially report that PLGA microspheres, which have been widely used as medicine-delivering carriers, were used to prepare antigen-specific killer complexes and treat allograft rejection. Our data highlight the therapeutic potential of this biocompatible and biodegradable antigen-specific killer effector for the treatment of allograft rejection and autoimmune disease.     

The influence of allo-class II MHC-specific Th2 cells on the generation of CD4 and CD8 cytotoxic T cells to associated class I and class II MHC alloantigen

There is considerable interest in whether CD4 T cell function can affect the outcome of allogeneic transplants. In mice tolerant to an isolated class II MHC disparity, the normal Th1 activity in vitro associated with graft rejection is switched to Th2 in tolerant animals. Because clinical transplants involve multiple class I and II MHC disparities we tested how the switch to Th2 activity of tolerant mice would affect the generation of CD4 and CD8 cytotoxic T cells (CTL) against MHC alloantigens to which the mice were not tolerant. A.TH mice (K^kI^sD^d) were rendered neonatally tolerant of A.TL (K^kI^kD^d) and the generation of CD4 or CD8 CTL measured in a mixed lymphocyte reaction (MLR) against (A.TLxB6)F₁ stimulators. Normal mice generated CD4 CTL against both A.TL and B6 (K^bI^bD^b), but tolerant mice were unable to generate cytotoxicity against either A.TL or B6. However, tolerant cells were able to generate CD8 CTL against B6. IL-4 inhibited the generation of CD4, but not CD8, CTL by normal cells and anti-IL-4 antibody was shown to increase the generation of CD4 CTL against B6 in F₁ stimulated cultures. Overall the results showed that a Th2 response could inhibit the generation of CD4 CTL against concomitant alloantigen in a process at least partially involving IL-4, but that, conversely, tolerant Th2 cells could help in the generation of CD8 CTL. The results suggest that with whole MHC disparities a simple change of CD4 T cells to Th2 would not be enough to procure graft acceptance.     

Dual effects of the alloresponse by Th1 and Th2 cells on acute and chronic rejection of allotransplants

The contribution of direct and indirect alloresponses by CD4⁺ Th1 and Th2 cells in acute and chronic rejection of allogeneic transplants remains unclear. In the present study, we addressed this question using a transplant model in a single MHC class I‐disparate donor–recipient mouse combination. BALB/c‐dm2 (dm2) mutant mice do not express MHC class I L^d molecules and reject acutely L^d+ skin grafts from BALB/c mice. In contrast, BALB/c hearts placed in dm2 mice are permanently accepted in the absence of chronic allograft vasculopathy. In this model, CD4⁺ T cells are activated following recognition of a donor MHC class I determinant, L^d 61–80, presented by MHC Class II A^d molecules on donor and recipient APC. Pre‐transplantation of recipients with L^d 61–80 peptide emulsified in complete Freund's adjuvant induced a Th1 response, which accelerated the rejection of skin allografts, but it had no effect on cardiac transplants. In contrast, induction of a Th2 response to the same peptide abrogated the CD8⁺ cytotoxic T cells response and markedly delayed the rejection of skin allografts while it induced de novo chronic rejection of heart transplants. This shows that Th2 cells activated via indirect allorecognition can exert dual effects on acute and chronic rejection of allogeneic transplants.     

Selection of T-cell receptors with a recurrent CDR3β peptide-contact motif within the repertoire of alloreactive CD8(+) T cells

Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo‐rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H‐2K^b (K^b)‐associated peptides and critically required to induce full activation of H‐2^k monoclonal CD8⁺ TLs expressing the cognate TCR‐BM3.3. Here, we asked whether a pBM1/K^b‐specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K^b‐binding CD8⁺ TLs increased from <0.04% in naïve mice to 3% of activated CD44⁺ CD8⁺ TLs in H‐2^k mice rejecting K^b‐expressing cells. Among these, TCR‐Vβ2 usage was greatly enriched, and 75% of them shared a TCR‐Vβ2 CDR3β motif with the prototype TCR‐BM3.3. Fewer than 5% of K^b‐reactive CD44⁺ CD8⁺ TLs not binding pBM1/K^b displayed this CDR3β motif. We found that the recurrent CDR3β motif of pBM1/K^b‐binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR‐peptide/MHC fit. Thus, a CDR3β motif generated by a process akin to “convergent recombination” accounts for a sizable fraction of the alloreactive anti‐K^b TCR repertoire.     

Failure to induce organ-specific autoimmunity by breaking of tolerance: importance of the microenvironment

Peripheral tolerance is considered to be a safeguard against autoimmunity. Using a TCR-transgenic mouse system displaying peripheral tolerance against a liver-specific MHC class I K^b antigen, we investigated whether the breaking of tolerance would result in autoimmunity. Reversal of tolerance was achieved by simultaneous challenge with cells expressing the K^b autoantigen and IL-2. Tolerance could not be broken with IL-2 alone or when K^b- and IL-2-expressing cells were applied to different sites of the mice. However, despite the presence of activated autoreactive T cells that were able to reject K^b-positive grafts no autoaggression against the K^b-positive liver was observed. These results indicate that breaking of tolerance per se is not sufficient to cause liver-specific autoimmunity. However, when in addition to breaking tolerance the mice were infected with a liver-specific pathogen, autoaggression occurred. Thus, in this system at least two independent steps seem to be required for organ-specific autoimmunity: reversal of peripheral tolerance resulting in functional activation of autoreactive T cells and conditioning of the liver microenvironment which enables the activated T cells to cause tissue damage.     

Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H-2Kb in the liver

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H-2K^b (K^b) under the control of the sheep metallothionein promoter (Met-K^b mice). To follow the fate of K^b-specific T cells, and to prevent any aberrant thymic expression of the K^b transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a K^b-specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non-transgenic thymus grafts. Although K^b-specific CD8⁺ T cells were produced in the thymus grafts of these manipulated Met-K^b mice, only small numbers of such cells could be detected in the spleen and lymph nodes. The livers, however, showed signs of damage and were heavily infiltrated by actively dividing CD8⁺ T cells. We provide strong evidence that the hepatocytes, not generally regarded as antigen-presenting cells, activated the K^b-specific CD8⁺ T cells and that these disappeared after a vigorous autoimmune response that resulted in deletion.     

Deletion of alloantigen-reactive thymocytes as a mechanism of adult tolerance induction following intrathymic antigen administration

Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2^b/BL10) alloantigens can be induced in CBA/Ca (H2^k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4⁺ T cells using a depleting anti-CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2^k) expressing a CD8-independent TCR specific for H2K^b were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 × 10⁷ BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2K^b-specific deletion of transgenic-TCR⁺ (tg-TCR) thymocytes which was dependent on the level of tg-TCR expression. IT deletion and the failure to export CD8⁺ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2K^b; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2K^b+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8⁺ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR-transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2K^b following transplantation.     

Differential expression of CLIP: MHC class II and conventional endogenous peptide: MHC class II complexes by thymic epithelial cells and peripheral antigen-presenting cells

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide : MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of Eα52–68: I-A^b and class II-associated invariant chain peptide (CLIP): I-A^b complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of Eα52–68:I-A^b complexes in thymic epithelial cells is approximately 5–10 % of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A^b complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.     

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I,but not class II molecules

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semi-syngeneic and MHC-matched bone marrow grafts was investigated. The use of β₂-microglobulin (β₂m) -/- and β₂m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of β₂m did not allow H-2^b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2D^d transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of β₂m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β₂m led to loss of the capability to reject H-2-matched β₂m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Tolerance induction as a multi-step process

Tolerant T cells are characterized by their partial or full resistance to activation by antigen. We investigated whether tolerant T cells were still receptive to further tolerogenic signals. T cells expressing a transgenic T cell receptor (TCR) specific for the major histocompatibility complex (MHC) class I molecule K^b were deleted in mice carrying K^b but not in mice expressing the mutant K^b-molecule K^bm1 [TCR (H-2^{bm1 × k}) mice]. These T cells were tolerant in vivo but could be activated in vitro by the K^b antigen. This in vitro reactivity was abolished after the tolerant T cells encountered K^b-positive cells that had been intravenously injected. Furthermore, in TCR (H-2^{bm1 × k}), mice expressing K^b only on hepatocytes, no T lymphocytes bearing the transgenic TCR could be found in the periphery, indicating that the additional contact with K^b on hepatocytes led to deletion of the tolerant T cells. These findings demonstrate that tolerance induction can be a multi-step process.     

Calnexin expression does not enhance the generation of MHC class I-peptide complexes

We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, K ^d , K^b , K^k , D ^d , D^b , and L^d . Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For K^b and K ^d , the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse β2-microglobulin. Also addressed was the effect of calnexin on the surface expression of K^b molecules bearing the immunodominant peptide from ovalbumin (OVA_{257 – 264} ). This was detected with a recently described monoclonal antibody specific for the K^b/peptide complex. Calnexin expression had no significant effect on the formation of K^b /peptide complexes generated from full-length OVA, cytosolic OVA_{257 – 264} , or endoplasmic reticulum-targeted OVA_{257 – 264} , which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of K ^d /peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.