Absence Of Alloantigen Research Articles

T Lymphocyte-Specific Activation of Nrf2 Protects from AKI.

T lymphocytes are established mediators of ischemia reperfusion (IR)-induced AKI, but traditional immune principles do not explain their mechanism of early action in the absence of alloantigen. Nrf2 is a transcription factor that is crucial for cytoprotective gene expression and is generally thought to have a key role in dampening IR-induced AKI through protective effects on epithelial cells. We proposed an alternative hypothesis that augmentation of Nrf2 in T cells is essential to mitigate oxidative stress during IR-induced AKI. We therefore generated mice with genetically amplified levels of Nrf2 specifically in T cells and examined the effect on antioxidant gene expression, T cell activation, cytokine production, and IR-induced AKI. T cell-specific augmentation of Nrf2 significantly increased baseline antioxidant gene expression. These mice had a high frequency of intrarenal CD25(+)Foxp3(+) regulatory T cells and decreased frequencies of CD11b(+)CD11c(+) and F4/80(+) cells. Intracellular levels of TNF-α, IFN-γ, and IL-17 were significantly lower in CD4(+) T cells with high Nrf2 expression. Mice with increased T cell expression of Nrf2 were significantly protected from functional and histologic consequences of AKI. Furthermore, adoptive transfer of high-Nrf2 T cells protected wild-type mice from IR injury and significantly improved their survival. These data demonstrate that T cell-specific activation of Nrf2 protects from IR-induced AKI, revealing a novel mechanism of tissue protection during acute injury responses.

Requirements for Prolongation of Allograft Survival with Regulatory T Cell Infusion in Lymphosufficient Hosts

For the clinical applicability of regulatory T cells (Tregs) in transplantation, it is critical to determine if donor antigen specificity is required for their immunosuppressive function. We developed an allospecific CD4(+) T cell receptor transgenic (TCR-tg) mouse as a source for large numbers of Tregs with defined allospecificity and tested whether they are more effective than polyclonal Tregs at suppressing allograft rejection. CD4(+)CD25(+)CD62L(hi) T cells were sorted from the spleen and peripheral lymph nodes of wild-type (WT-Tregs) and TCR-tg (Allo-Tregs) mice, and expanded using IL-2 and anti-CD3/anti-CD28 conjugated magnetic beads. Tregs were tested for their ability to suppress the proliferation and cytokine production of alloreactive CD4(+)CD25(-) T cells in mixed leukocyte assays. Syngeneic WT hosts were adoptively transferred 5 × 10(6) Tregs and transplanted with allogeneic hearts. Using anti-CD3/anti-CD28 conjugated beads, Tregs were expanded in vitro 100-fold and maintained their suppressor phenotype and function. Allo-Tregs were 6-8 times more potent on a cell-for-cell basis than WT-Tregs in suppressing allospecific proliferation in vitro. Allo-Tregs were unable to suppress in the absence of allo-antigen. Adoptive transfer of expanded Allo-Tregs into WT recipients prolonged the graft survival in a F1 heart transplant model compared with WT-Treg or no treatment [20.0 ± 4.4 d (n = 6) versus 10.4 ± 1.2 (n = 8) and 9.7 ± 1.6 d (n = 6)]. Unlike polyclonal Tregs, allospecific Tregs are able to prolong allograft survival. However, large numbers of Allo-Tregs were unable to induce tolerance, suggesting that Treg therapy in immunocompetent recipients will require conditioning and/or additional immunomodulation for the induction of tolerance.

A role for regulatory T cells in renal acute kidney injury

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4 +CTLA4 +Foxp3 + cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4 +CTLA4 +Foxp3 + cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1(anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10 ± 50.04; PC61: 187.23 ± 31.38; DTA-1: 64.53 ± 25.65, mg/dL, p < 0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61(anti-CD25) mAb treatment is deleterious, while DTA-1(anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI.

Very early alloantigen-independent trafficking of lymphocytes during ischemic acute kidney injury

Lymphocytes play an important role during ischemia-reperfusion injury (IRI). Lai et al. have demonstrated, for the first time, an increase in kidney lymphocytes 1 hour after IRI, a newly identified kidney lymphocyte reservoir, and have confirmed the pathogenic role of lymphocytes by manipulating the sphingosine-1-phosphate (SIP)-sphingosine-1-phosphate type 1 (S1P1) receptor pathway.

Graft-Versus-Tumor (GvT) Effects in the Absence of Alloantigens on Tumor Cells Crucially Depend on Graft-Versus-Host Disease (GvHD) Intensity.

While a variety of tumors is potentially immunogenic and is capable to trigger tumor-associated antigen (TAA)-specific immune responses in vivo the endogenous immune system usually fails to eradicate established tumors. Despite considerable effort and promising experimental and preliminary clinical data most cellular immunotherapeutic strategies have not evolved into a clinically relevant treatment strategy with allogeneic immune replacement as cancer immunotherapy being the sole exception. At this juncture allogeneic HSCT not only allows for otherwise fatal high-dose chemo- and radiotherapy intended to reduce the tumor cell burden but also encompasses potent immunologically mediated anti-tumor effects, referred to as GvT reaction. This GvT reaction, which is based on donor T and natural killer cells, is interrelated, however, with potentially deleterious GvHD, one of the major limitations of allogeneic HSCT. Mutated tumor-associated antigens, as well as selectively or aberrantly expressed nonmutated antigens, represent potential targets for T cell-mediated GvT effects that are in principle, separable from generalized anti-host responses. Using an allogeneic parent-into-F1 murine transplantation model (BALB/c or C57BL/6 → [C57BL/6 x BALB/c]F1) with different chemically induced tumors derived from either parental strain, we have previously demonstrated that a significant CD8+ T cell-mediated GvT effect accounts for immunological tumor control after allogeneic HSCT in vivo. Remarkably, this effect occurs in the absence of histoincompatibilities between donor immune cells and host tumor (Blood 2004; 104:1210–1216). Since most chemically induced tumors have immunogeneic neo-antigens we sought to expand our initial findings to spontaneously occurring tumor cell lines, which often are less immunogenic. In addition, we asked whether the intensity of GvHD directly impacts on GvT effects in the absence of alloantigens on tumor cells. Sublethally irradiated [C57BL/6 x BALB/c]F1 (H-2b/d) mice were transplanted with whole bone marrow with or without splenocytes from allogeneic C57BL/6 (H-2b) and BALB/c (H-2d) donors, respectively. Recipient mice were inoculated subcutaneously with hematological and solid tumors derived from either C57BL/6 (B16, LLC - both H-2b) or BALB/c (RENCA, A20, MPC-11, WEHI-3B - all H-2d) strains. By varying the histocompatibility between donor immune cells, host tissue and tumor tissue, it was possible to study GvT effects in the absence of alloantigens on tumor cells. Furthermore, using different numbers of coinjected donor splenocytes the interrelation of GvHD intensity and alloantigen independent GVT effects was assessed. As compared with allograft recipients having weak GvHD (2x107 bone marrow cells) a significant tumor-alloantigen independent GvT effect was present in animals undergoing intense GvHD (2x107 bone marrow cells and 107 splenocytes). These data indicate that a biologically relevant GvT effect that is independent of alloantigen recognition on tumor cells is crucially influenced by the intensity of GvHD.

The Mechanism of Transfusion-Related Immunomodulation Is Related to the Transfusion of Dendritic Cells Expressing the CD200 (OX-2) Tolerance Signal and Alloantigen: Evidence from a Murine Tumor Model.

Background: The transfusion of blood products containing allogeneic leukocytes can alter recipient resistance to infection and stimulate the growth of some types of tumors in animal models of transfusion-induced tumor growth. Transfusion related immunomodulation (TRIM) represents a credible mechanism for the altered resistance to infection seen clinically, but cannot explain the growth of non-immunogenic tumors in syngeneic hosts seen regularly in the various transfusion-related animal models of tumor growth.Methods and Results: In this study the transfusion of 50–200 μL of unmodified anticoagulated allogeneic BALB/c (H-2d) mouse blood into C57B1/6 (H-2b) mice, four days before being intravenously injected with syngeneic (H-2b) FSL10 fibrosarcoma cells (1–2 x 106 cells per mouse) resulted in a significant increase in the number of pulmonary nodules observed at 3 weeks compared to that seen in control mice. The median number of pulmonary nodules increased in an allogeneic blood transfusion dose-dependent manner, as did the proportion of mice without pulmonary nodules. This tumor growth-promoting effect of the allogeneic blood transfusions required the presence in the transfused blood of allogeneic CD11c+ dendritic cells bearing the CD200 co-stimulatory tolerance signal. This tumor growth-promoting effect of allogeneic blood could be blocked by specific monoclonal antibodies to either CD11c or to CD200. CD200 receptor-mediated signaling alone, in the absence of alloantigen, failed to augment the number of TRIM-induced pulmonary tumor nodules. Physiological concentrations of TGFβ, but not IL-10, were shown to stimulate proliferation of FSL10 cells in vitro in these studies. In this context, it is known that CD200 together with alloantigens are known to stimulate development of suppressor cells acting via IL-10 and TGFβ in vivo. Allogeneic blood also caused a significant CD200-dependent accumulation of TGFβ+ suppressor cells in the spleen, 12 days after transfusion, when the spleen cells could be shown to adoptively transfer the TRIM effect to naive animals.Conclusions: These data support the hypothesis that allogeneic transfusions in an allogeneic blood transfusion mouse tumor model results in tumor growth promotion in recipient mice. This effect appears to result in both the induction of TGFβ-producing suppressor cells as well as requiring the transfusion of allogeneic CD11c+ dendritic cells, bearing both CD200 tolerance signaling molecules and alloantigens.

Initial Inhibition of Tissue Factor Signalling Reduces Chronic Vascular Changes in Isogenic Rat Aortic Transplants

Vascular changes are considered the major histopathological indicator of chronic allograft dysfunction. These changes are characterized by intimal thickening caused by accumulation of primarily smooth muscle cells. Contributing factors may be of both immunological and nonimmunological origin. Cold ischemia has been shown to trigger intimal proliferation in the absence of alloantigen in an isogenic rat aortic transplant model. We have used this model to investigate the impact of inhibition of tissue factor (TF) signalling on the progression of intimal thickening. Group 1 was treated with recombinant FVIIa inhibited in its active site (rFVIIai), and group 2 served as untreated controls. At 8 weeks the intimal area was measured with image analysis. Medial areas and the proportion of medial necrosis were determined. Animals treated with rFVIIai showed significantly less intimal thickening compared with controls: median 0.147 vs. 0.256 mm2, respectively (p = 0.008). A positive correlation between intimal hyperplasia and medial necrosis (rs = 0.79, p = 0.01), as well as adventitial inflammation (rs = 0.83, p = 0.009), was found. TF mRNA was not detected in the neointima at 8 weeks, as determined by in situ hybridization.We conclude that active site inhibited FVIIa (rFVIIai) given prior to and directly after implantation of aortic transplants significantly reduces intimal hyperplasia caused by nonimmunological factors in this model.

CD28-B7 blockade in organ dysfunction secondary to cold ischemia/reperfusion injury: Rapid Communication

Ischemic injury to cadaver organs is a major risk factor for development of chronic organ dysfunction. We have recently shown that the B7 costimulatory pathway plays a critical role in early organ dysfunction developing after renal cold ischemia/reperfusion injury. We extended these observations to investigate the role of this pathway in the development and progression of chronic organ dysfunction following such injury. Uninephrectomized rats which underwent cold ischemia/reperfusion injury developed progressive proteinuria as compared to uninephrectomized controls. Animals treated with CTLA4Ig, which blocks B7 costimulation, starting on the day of injury had significantly better long-term survival and developed significantly less proteinuria than control animals treated with control Ig. RT-PCR analysis of kidney tissue showed significant reduction in expression of activation and inflammatory cytokines, chemoattractants, and growth factors, as compared to controls. Delaying administration of CTLA4Ig for one week, but not four weeks, after injury was still effective in ameliorating development of progressive proteinuria. Interestingly, selective blockade of B7-1 by a mutant form of CTLA4Ig had no effect on early or chronic organ dysfunction. These findings indicate the long-term functional and molecular consequences of experimental cold ischemia/reperfusion injury, and suggest that B7-2 is critical in the development of organ dysfunction following ischemic injury, even in the absence of alloantigen.

The role of the B7 costimulatory pathway in experimental cold ischemia/reperfusion injury.

Ischemia/reperfusion injury associated with organ retrieval and storage influences the development of chronic graft dysfunction, the major clinical problem in solid organ transplantation. The potential role of mononuclear cells (T cells and monocyte/macrophages) in this type of injury is unknown. Inbred male Lewis rats were uninephrectomized and the left kidney perfused in situ with 10 ml of iced University of Wisconsin solution. Immunohistological studies showed mononuclear cell infiltration of the ischemic organs associated with the upregulation of MHC class II antigen expression. Reverse transcriptase-PCR indicated that T cell associated cytokines and monocyte/macrophage activation markers/products are upregulated early after the ischemic insult. B7 expression occurred within 24 h and peaked at 3 d. Plasma creatinine levels rose transiently with complete recovery of renal function by 5 d. Animals began to develop progressive proteinuria after 8-12 wk, indicative of the long-term functional consequences of early ischemia/reperfusion injury. Blockade of T cell CD28-B7 costimulation with CTLA4Ig resulted in significant inhibition of T cell and macrophage infiltration and activation in situ. Treated animals did not exhibit transient renal dysfunction, nor developed proteinuria over time. This is the first demonstration that blocking T cell costimulatory activation in the absence of alloantigen can prevent the early and late consequences of ischemia/reperfusion injury.

SICAM-1 Enhances Cytokine Production Stimulated by Alloantigen

The leucocyte adhesion molecules lymphocyte functional antigen-1 (LFA-1; CD11a/CD18) and intercellular adhesion molecule-1 (ICAM-1; CD54) facilitate the cell-to-cell interactions which are required for the initiation of immune responses. The role of this interaction in the response to alloantigen was assessed by comparing the effects of monoclonal antibodies against these molecules to the effects of a soluble form of the ICAM-1 molecule in the mixed lymphocyte response (MLR). In contrast to the well-documented inhibitory effects of anti-ICAM-1 or anti-LFA-1 antibodies on mixed lymphocyte responses, we were unable to block these responses with the soluble form of ICAM-1 (sICAM-1). In contrast, the addition of sICAM-1 to these cultures resulted in a two- to sixfold enhancement in the T-cell proliferative response to alloantigen over the normal response. Unlike previous reports, the biological activity of sICAM-1 was not dependent on generation of a solid-phase form of the molecule. The enhanced proliferative response correlated with an increase in the level of TNF-α detected in the MLR supernatants and could be blocked by antibodies to TNF-α. sICAM-1 had no effect on proliferation or cytokine production in the absence of alloantigen. These results suggest that antibodies which block ICAM-1/LFA-1 not only block the adhesion which is required to stabilize cell-to-cell contact, but also block the costimulatory signal which is required for T-cell activation.

Rapid re‐expression of CD45RC on rat CD4 T cells in vitro correlates with a change in function

Rat CD4+ T cells are divided phenotypically by the anti-CD45RC monoclonal antibody OX22 into subsets with contrasting functions. Stimulation of T cells in vitro is known to induce a change in isoform from CD45RC+ to CD45RC-. We have investigated the in vitro conditions which promote a switch in isoform in the opposite direction. We observed that a majority of CD45RC- CD4 T cells (> 90%) spontaneously re-expressed CD45RC during the first 1-3 days of culture in both the presence and absence of alloantigen. The T cells remained CD45RC+ when cultured for 7 days in serum-free growth medium. However, alloantigen-activated lymphocytes, expressing the interleukin-2 receptor (IL-2R), downregulated CD45RC by day 4 and remained CD45RC- during the course of the experiment. Using mixtures of allotype-marked CD45RC+ and CD45RC- T cells, it was demonstrated that each subset showed comparable survival, IL-2R expression and time courses of activation in response to alloantigen. The repertoire of neither subset was, therefore, deficient in terms of allorecognition. The rapid re-expression of CD45RC in culture was accompanied by a change in function: CD45RC+ "converts", obtained by overnight culture of CD45RC- T cells, induced significantly higher graft-versus-host responses. Thus, the transition in culture from CD45RC- to CD45RC+ reflects a major functional reprogramming of the cell and not a trivial modulation of a surface antigen.

IL-12 augments antigen-dependent proliferation of activated T lymphocytes.

Ag-dependent T cell activation requires multiple transmembrane signals including activation of Ag-specific T cell receptor in combination with signals delivered through cytokine receptors. IL-12 is a heterodimeric cytokine involved in the regulation of NK and T lymphocyte responses. In examining the role of IL-12 in T cell activation, we found a direct relationship between Ag stimulation and IL-12-induced proliferation. Unlike IL-2, which induced proliferation of CTL either in the presence or absence of a CD3/TCR co-signal, IL-12 mediated proliferation of CTL only when the cells were recently co-stimulated with alloantigen or solid-phase anti-CD3 antibody. After culture in the absence of alloantigen or anti-CD3 for 7 to 14 days, these CTL lost the ability to proliferate to IL-12 alone. Under these conditions, however, IL-12 synergized with low-dose IL-2 to induce CTL proliferation. Restimulation with alloantigen or solid-phase anti-CD3 restored the ability of the CTL to proliferate to IL-12 alone. Not all Ag signals resulted in IL-2 independent proliferation to IL-12. For example, CTL with specificity for influenza matrix peptide proliferated best when co-cultured with peptide Ag presented on self MHC and a combination of IL-2 and IL-12. This evidence suggests that IL-12 may be useful in expanding an Ag-specific T cell population, as the culture of CTL with IL-12 and low-dose IL-2 leads to proliferation only in response to an Ag co-signal.

Accessory cell-T lymphocyte interactions. Antigen-dependent and -independent clustering.

Previous work documented the capacity of dendritic cells (DC) to stimulate primary immune responses and to physically cluster with the responding lymphocytes. Rapid cell-cell aggregation assays were used here to study the interaction of DC and other types of APC with T lymphocytes. Graded doses of APC were sedimented with T cells that had been primed to alloantigens, soluble proteins, or lectin, and then labeled with carboxyfluorescein diacetate. The number of clustered T cells was measured after 10 min at 4 or 37 degrees C. At 4 degrees, binding was antigen-dependent and included greater than 50% of the added T cells. Clustering was mediated by all types of APC tested, including DC, macrophages, B lymphocytes, and fresh Langerhans cells, although DC were the most effective. Specificity was evident in the findings that alloreactive T lymphoblasts bound to allogeneic but not syngeneic APC; KLH- and OVA-reactive T cells bound to syngeneic APC in the presence of specific protein: and Con A blasts needed lectin to cluster. A 30 min pretreatment with chloroquine, a drug known to inhibit APC activity, markedly blocked the specific binding of alloreactive and protein-specific T blasts at 4 degrees C. Since Lyt-2- alloreactive blasts should specifically recognize Ia, presentation of Ia seems to be altered by chloroquine. Binding assays at 37 degrees C gave similar results to those performed at 4 degrees C, with one exception. When DC were used as APC, striking antigen-independent clustering occurred. DC could efficiently cluster primed T cells in the absence of alloantigen, soluble protein, or lectin. We suggest that antigen-independent binding contributes to the distinctive capacity of DC to prime T cells in the afferent limb of the immune response, whereas antigen-dependent binding between other APC and sensitized lymphocytes is critical in the efferent limb.

Mixed lymphocyte reaction suppression by tumour cell lines from naturally occurring squamous cell carcinomata.

Seven epithelial tumour cell lines derived from naturally occurring ovine squamous cell carcinomata (OSCC) were examined for their ability to stimulate allogeneic lymphocytes in vitro. All cell lines failed to stimulate any proliferative response; a failure which was not due to the absence of alloantigens on the tumour cells. It was found, however, that the cell lines tested caused a marked suppression of a third party mixed lymphocyte reaction. This suppression was mediated only by live tumour cells since the addition of heat-killed tumour cells had no effect on the response.

Histamine production by alloantigen-activated mouse bone marrow cells

Histamine's contribution to the manifestations associated with graft-versus-host disease (GVHD) and/or hybrid resistance is unknown. Thus, we initiated studies to see whether or not mouse bone marrow cells could produce histamine upon alloantigen stimulation. Irradiated allogeneic spleen cells were shown to stimulate bone marrow cells to produce and secrete high levels of histamine. During 7 days of culture there was only a marginal increase in cell-associated histamine while the amount of histamine in the supernatant increased 10- to 20-fold. Optimal histamine production was dependent upon Lyt 1 +2 + T cells resident in the bone marrow. Further, bone marrow cells from Nude mice failed to produce high levels of histamine following alloantigen stimulation. Soluble factors produced by alloantigen-stimulated bone marrow cells or by Con A-stimulated rat spleen cells induced high levels of histamine production in bone marrow cells in the absence of alloantigen. We suggest that histamine production by alloantigen-activated bone marrow cells may modulate immune functions following bone marrow transplantation.

Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells.

A synergistic interaction in the proliferative response to alloantigen has been previously noted when intact thymus cells are cultured with post-thymic (peripheral) lymphoid cells. In the present study, a factor extracted from the thymus has been shown to similarly enhance the reactivity of syngeneic lymph node cells and thus to retain the amplifier activity of intact thymus cells. The factor has no effect on lymphoid cell proliferation in the absence of alloantigen. Cells with amplifier activity are found in highest concentration in the thymus but also may be detected in spleen cells that are nonadherent to nylon wool. The factor is shown in these experiments to be derived from thymic lymphoid cells and to act primarily upon post-thymic (peripheral) lymphoid cells. As such, this factor appears to be distinct from various other thymus factors that have been localized to thymic reticuloepithelial elements and that are thought to effect predominantly the differentiation of T-cell precursors.

Minor H Antigens Introduced on H-2 Different Stimulating Cells Cross-React at the Cytotoxic T Cell Level during in Vivo Priming

The cellular basis of the cross-priming observed with minor histocompatibility antigens on H-2 different cells was investigated. Cytotoxic T cells induced against minor alloantigens show absolute H-2 restriction at the effector level ([51Cr]-release). That is, F1 (BALB/c X BALB.B) (H-2d/b) cytotoxic cells induced by immunization with B10(H-2b) cells are not able to lyse B10.D2(H-2d) targets. But an injection of B10 cells does prime F1 mice for a secondary cytotoxic response to B10.D2. The technique of inducing cytotoxic effector function polyclonally with Con A in the absence of alloantigen was used here to establish that such cross-priming reflects what happens at the cytotoxic cell level. It is shown that an F1 animal previously injected with B10 cells has expanded pools of memory cytotoxic cells reactive with B10 and B10.D2. From this it is concluded that: a) minor H structures on B10.D2 and B10 do cross-react at the cytotoxic T cell level during in vivo priming, and b) because normal cells cross-prime whereas tumor cells do not, then the F1 cytotoxic precursors are probably committed to respond to antigen on cells bearing either the maternal or paternal H-2 haplotype before they encounter antigen. Cross-priming may be explained by foreign minor H antigens being presented to F1 host T cells on the surface of host macrophages. Therefore priming is not restricted to the H-2 type of the injected cells but to both H-2 types of the F1 host.