2C CD8 Research Articles

Calcineurin Inhibitors Inhibit Tolerance Induction By Suppressing Terminal Differentiation of Donor Exhausted T Cells after Allogeneic SCT

[Introduction] Calcineurin inhibitors (CNIs) reduce acute GVHD by inhibiting NFAT signaling in donor T cells but fail to prevent chronic GVHD (cGVHD). We have previously shown that donor alloreactive T cells differentiated into exhausted T cells with severely impaired alloreactivity after mouse allogeneic hematopoietic stem cell transplantation (allo-SCT) (Asakura S: JCI 2010). Given the critical role of NFAT signaling in T-cell exhaustion, we explored the role of CNIs in donor T-cell exhaustion and tolerance induction after allo-SCT using mouse SCT models with host alloantigen (allo-Ag)-specific 2C TCR-transgenic (2C) T cells and single cell RNA sequencing (scRNAseq) of donor T cells. [Methods] B6D2F1 recipients (H-2b/d) were lethally irradiated and transplanted with 1 x 106 2C T cells (CD8.1+) plus 1 x 106 wild type T cells (WT, CD45.1+) of B6 background (H-2b) together with T-cell-depleted bone marrow cells (TCD-BM) from WT B6 (CD45.2+) on day 0. In some experiments, recipients were p.o. injected with 25 mg/kg cyclosporin (CSP) daily on days 0-14. [Results] Flowcytometric analysis (FCM) showed 2C CD8+ T cells differentiated into terminally exhausted T cells (Tex) expressing multiple co-inhibitory receptors such as PD-1 and TIGIT on day 14 after allo-SCT, that led to unresponsiveness of donor T cells to PD-1 blockade. Next, we performed scRNAseq of donor T cells harvested from CSP- or vehicle-treated recipients on day 14 after allo-SCT. UMAP analysis identified 4 clusters of CD4+ T cells (C10-C13) and 9 clusters of CD8+ T cells (C1-C9), and all of them expressed Tox and Pdcd1, indicating that all donor T cells entered differentiation pathway to exhaustion. CSP remarkably increased C10 (CD4+) and C1 (CD8+) characterized by lower expression of Tox and Pdcd1 and higher expression of Ly6c2 compared to other clusters (Fig 1). FCM confirmed CSP significantly expanded PD-1+TIGIT-, and TOXlow cells in 2C and WT CD8+ and WT CD4+ donor T cells on day 14, and these cells persisted at least 2 wks after cessation of CSP. After adoptively transferred into irradiated secondary B6D2F1 recipients, Ly6C+ T cells purified from CSP-treated recipients were differentiated into Ly6C- Tex, while Ly6C- T cells remained to be Ly6C-, indicating that CSP-induced Ly6C+ donor T cells are precursors of Tex (pTex) like cells. We named these cells as pTex-CNI, based on lower TCF-1 expression compared to TCF-1high canonical pTex. We found that, among various agents known to suppress GVHD or Tex differentiation, only CSP and ibrutinib induced ToxlowPD-1+TIGIT-Ly6C+ pTex-CNI, suggesting that NFAT inhibition is critical for pTex-CNI induction. Importantly, only Ly6C+ donor T cells, but not Ly6C- cells, induced cGVHD after adoptive transfer, suggesting that pTex-CNI play a critical role in pathophysiology of cGVHD (Fig 2). pTex-CNI demonstrated marked proliferative response and differentiation toward Tex upon PD-1 blockade as canonical pTex did in tumor and viral infection models in the previous reports (Sade-Feldman M: Cell 2018, Hudson WH: Immunity 2019). Administration of PD-L1 mAbs from day 14 enhanced OXPHOS of donor T cells and eradicated host-type leukemia cells only in CSP-treated recipients. Post-transplant cyclophosphamide (PTCy) is widely used in clinical allo-SCT, and CNIs are started either before SCT or after PTCy typically on day 5. We found that pTex-CNI were induced only when CSP was added to PTCy from day -1, but not from day 5, suggesting that NFAT inhibition early after allo-SCT is critical for induction of pTex-CNI. Finally, we confirmed that T cells from the patients who underwent HLA-matched PBSCT with tacrolimus (TAC)-based GVHD prophylaxis started from day -1 expressed significantly lower levels of PD-1, TIGIT, and TOX on day 28 compared to those from patients underwent haploidentical PBSCT with PTCy-based GVHD prophylaxis combined with TAC started from day 5. [Conclusion] Our study for the first time demonstrated that CNI inhibits terminal differentiation of donor exhausted T cells after allo-SCT. In mouse models, pTex-CNI contributed to development of cGVHD and PD-1 blockade-induced anti-tumor response after allo-SCT. The timing of CNI initiation in PTCy-based allo-SCT could determine the persistence of donor T cell alloreactivity after allo-SCT, that could impact on the strategies of cessation of immunosuppressants and treatment of relapsed diseases. These should be tested in the future clinical studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The fate of leukemia-specific CD8+ T cell clones is dictated by antigen specificity and the mechanism of cognate antigen presentation

Abstract We previously demonstrated that high-affinity leukemia-specific CD8+ T cells are deleted in leukemia-bearing hosts in a manner that depends on cross-presentation of the antigen by splenic Batf3-lineage dendritic cells. Here, we generated a T cell receptor transgenic mouse strain (Tg101) that expresses TCR-α and -β chains from a CD8+ T cell clone specific for a native H-2Kb-restricted antigen on C1498 leukemia cells. In naïve mice, Tg101 T cells demonstrate no evidence of self-reactivity, arguing that the Tg101 antigen is leukemia-specific. In leukemia-bearing mice, high-affinity 2C CD8+ T cells specific for the H-2Kb-restricted model antigen, SIYRYYGL, encounter antigen rapidly (day 3–4) via its cross-presentation by splenic CD8a+ DCs, proliferate briefly, and are largely deleted by day 12. Conversely, antigen encounter by Tg101 CD8+ T cells in mice with leukemia is delayed (day 8–10), and primarily occurs in the liver - a prominent site of leukemia progression. Subsequently, Tg101 T cells expand, upregulate expression of co-inhibitory receptors, including PD-1, LAG-3, TIM-3 and TIGIT, and produce effector cytokines poorly, consistent with the acquisition of a dysfunctional state. Surprisingly, in vivo antigen encounter by Tg101 T cells occurs independently of CD11c+ cells, indicating that the Tg101 antigen is poorly cross-presented. Rather, the antigen is directly presented to Tg101 T cells by C1498 cells. Collectively, our data reveal that CD8+ T cell tolerance is effectively generated in leukemia-bearing animals. However, the nature of the leukemia antigen and the context in which it is presented are critical factors in dictating the underlying mechanism of tolerance that ensues. A search for the Tg101 antigen is ongoing.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Transfer of Allogeneic CD4+ T Cells Rescues CD8+ T Cells in Anti-PD-L1-Resistant Tumors Leading to Tumor Eradication.

Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T-cell-mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the "exhaustion" markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD-1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. Cancer Immunol Res; 5(2); 127-36. ©2017 AACR.

A Novel Pathway for CD8+ T Cell Activation by Donor Derived Non-Haematopoietic Cells Leading to Acute Allograft Rejection

Introduction: Although seminal studies have shown that CD8+ T cells can reject cardiac allografts that lack donor haematopoietic cells (HPCs) through a direct cognate interaction with graft parenchymal cells, the observation from other studies that secondary lymphoid tissue (SLT) is essential for graft rejection creates a paradox yet to be resolved. Here we examine the hypothesis that CD8+ T cell activation by graft parenchymal cells requires acquisition and re-presentation of intact MHC class I alloantigen by recipient antigen presenting cells (APCs) within SLT. If proven, this hypothesis would not only resolve this paradox, but would also represent the first definitive demonstration of the contribution of the semi-direct pathway to graft rejection. Methods: CD8+ T cell-mediated allograft rejection following activation by donor parenchymal cells was examined using a transgenic heterotopic murine heart transplant model. Balb/c cardiac donors were lethally irradiated and treated with depleting antibodies ensuring complete eradication of HPCs, such that parenchymal cells in these grafts were the only source of alloantigen presentation. These HPC-depleted Balb/c (Balb/cHPC-) cardiac allografts were transplanted into: 1) 2C transgenic mice (monoclonal population of CD8+ T cells against Ld MHC class I); 2) Splenectomised aly/aly (aly/alyspl) mice (complete absence of SLT) with adoptive transfer of 2C CD8+ T cells, to investigate the importance of SLT in this pathway; 3) CD11c-DTR transgenic mice, in which host dendritic cells (DCs) are selectively depleted by diphtheria toxin, allowing delineation of the role of these cells in parenchymal alloantigen presentation to CD8+ T cells. Results: 2C transgenic mice rejected Balb/cHPC- grafts as rapidly as non-depleted Balb/c grafts (MST = 5d vs. 4d respectively; p=0.4) suggesting an effective mechanism for parenchymal cell driven CD8+ T cell mediated rejection. Balb/cHPC- allografts showed prolonged survival (>50d) in aly/alyspl mice given 2C CD8+ T cells, whereas in non-splenectomised controls all grafts rejected (MST = 17d; p=0.01) suggesting an essential role for SLT in this pathway. In addition, when aly/alyspl mice were given activated 2C CD8+ T cells (from a 2C recipient of a Balb/c cardiac graft) they rapidly rejected Balb/cHPC- allografts (MST = 7d; p=0.01) indicating that SLT plays a fundamental role in the activation of CD8+ T cells. To examine, therefore, whether recipient DCs were required to present to CD8+ T cells within SLT, CD4+-depleted CD11c-DTR mice given 2C CD8+ T cells were transplanted with Balb/cHPC- allografts. When these mice were also treated with diphtheria toxin to deplete host DCs they demonstrated significantly delayed rejection kinetics when compared with untreated controls (MST = 26d vs. 16d; p=0.02) highlighting a key role for these cells. Conclusion: These results indicate that allorecognition of graft parenchymal cells represents an important mechanism of CD8+ T cell mediated rejection. They support the role of a novel pathway in which recipient DCs acquire intact MHC class I from donor parenchymal cells and present to CD8+ T cells in SLT leading to their activation.

Preterm labor leads to activation and proliferation of maternal T cells which recognize the paternal alloantigen via the indirect pathway (100.46)

Abstract Purpose: Multiple tolerance mechanisms protect the semi-allogeneic fetus from attack by the maternal adaptive immune system. We hypothesize preterm labor (PTL) may represent a breakdown of tolerance to the allogeneic fetus. Methods: B6 females were bred to BALB/c males and CFSE-labeled TCR transgenic T cells (TCR75 CD4, indirect; 4C CD4, direct; 2C CD8, direct) which recognize BALB/c antigen were adoptively transferred on E12.5 to study the roles of indirect and direct antigen presentation. PTL was induced by LPS on E13.5 and mice were analyzed at E18.5 for expression of CD44, CD62L, and CFSE. Controls included syngeneic matings with PTL and allogeneic matings without PTL. Results: 4C and 2C T cells did not proliferate during PTL. TCR75 cells proliferated and upregulated CD44 in allogeneic pregnancies with PTL compared to those without PTL (p<0.05) or syngeneic pregnancies with PTL (p<0.05). Although TCR75 cells proliferated in all allogeneic pregnancies, they only accumulated in animals undergoing PTL, suggesting that apoptosis of alloreactive T cells maintains maternal-fetal tolerance in normal pregnancies. Conclusions: PTL leads to proliferation, activation, and accumulation of maternal T cells that recognize the paternal alloantigen via the indirect pathway, indicating these cells participate in the pathogenesis of the disease. Understanding their role may uncover novel pathways for therapeutic intervention in PTL.

RasGRP1 Regulates Antigen-Induced Developmental Programming by Naive CD8 T Cells

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1(-/-) 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.

Direct Imaging of Immune Rejection and Memory Induction by Allogeneic Mesenchymal Stromal Cells

Although mesenchymal stromal cells (MSCs) exhibit marked immunoregulatory activity through multiple mechanisms, their potential to completely evade rejection upon transplantation into allogeneic recipients is controversial. To directly address this controversy, the survival of luciferase-labeled MSCs (Luc(+) MSCs) was evaluated by imaging in allogeneic recipients. This analysis showed that although MSCs exhibited longer survival compared to fibroblasts (Fib), their survival was significantly shorter compared to that exhibited in syngeneic or in immune-deficient Balb-Nude or non-obese diabetic severe combined immunodeficiency (NOD-SCID) recipients. Graft rejection in re-challenge experiments infusing Luc(+) Fib into mice, which had previously rejected Luc(+) MSCs, indicated potential induction of immune memory by the MSCs. This was further analyzed in T-cell antigen receptor (TCR) transgeneic mice in which either CD4 TEA mice or CD8 T cells (2C mice) bear a TCR transgene against a specific MHC I or MHC II, respectively. Thus, following a re-challenge with MSCs expressing the cognate MHC haplotype, an enhanced percentage of 2C CD8(+) or TEA CD4(+) T cells exhibited a memory phenotype (CD122(+), CD44(+), and CD62L(low)). Collectively, these results demonstrate that MSCs are not intrinsically immune-privileged, and under allogeneic settings, these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party ("off the shelf") MSCs is commonly advocated for a variety of clinical applications, our results strongly suggest that long-term survival of allogeneic MSCs likely represents a major challenge.

Recurrence of intracranial tumors following adoptive T cell therapy can be prevented by direct and indirect killing aided by high levels of tumor antigen cross-presented on stromal cells.

Elimination of peripheral tumors by adoptively transferred tumor-specific T cells may require killing of cancer cells and tumor stromal cells. Tumor Ags are cross-presented on stromal cells, resulting in direct cytotoxic T cell (CTL) killing of both Ag-expressing cancer cells and stromal cells. Indirect killing of Ag loss variant cells also occurs. We show here that similar processes occur in a brain tumor stromal environment. We used murine cancer cell lines that express high or low levels of a peptide Ag, SIYRYYGL (SIY), recognized by transgenic 2C CD8(+) T cells. The two cell lines are killed with equivalent efficiency by 2C T cells in vitro. Following adoptive transfer of 2C T cells into mice with established SIY-Hi or SIY-Lo brain tumors, tumors of both types regressed, but low-Ag-expressing tumors recurred. High-Ag-expressing tumors contained CD11b(+) cells cross-presenting SIY peptide and were completely eliminated by 2C T cells. To further test the role of cross-presentation, RAG1(-/-) H-2(b) mice were infused with H-2(k) tumor cells expressing high levels of SIY peptide. Adoptively transferred 2C T cells are able to kill cross-presenting H-2(b) stromal cells but not H-2(k) tumor cells. In peripheral models, this paradigm led to a small static tumor. In the brain, activated 2C T cells were able to kill cross-presenting CD11b(+) cells and completely eliminate the H-2(k) tumors in most mice. Targeting brain tumor stroma or increasing Ag shedding from tumor cells to enhance cross-presentation may improve the clinical success of T cell adoptive therapies.

Rapid Deletional Peripheral CD8 T Cell Tolerance Induced by Allogeneic Bone Marrow: Role of Donor Class II MHC and B Cells

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II(+) donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c(+) dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b(+) monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b(+) cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.

The Role of ERK and XIAP in Tolerance Induction by Veto CTLs.

Several bone marrow and lymphocyte subpopulations, known as ' veto cells' were shown to induce transplantation tolerance across major histocompatability antigens. We recently demonstrated, that allogeneic anti-3rd party CTLs depleted of alloreactivity against the host are endowed with marked veto activity and can facilitate allografting of T cell depleted bone marrow without GVHD. Earlier insights indicated that at low veto cell concentrations, both CD8 and FasL on the veto cell might be required to induce specific deletion of the effector cell. Such a mechanism involves initial recognition of the veto cell by the TCR of the effector cell, leading to expression of Fas upon activation of the latter and thereby allowing for Fas-FasL apoptosis. The presence of FasL on the veto cell cannot result in apoptosis of the effector cell unless CD8 on the veto cells is available and can interact with MHC class 1 on the effector cell. Thus, the CD8-MHC interaction seems to be associated with an increased susceptibility of the effector cell to FasL killing. Recently, by using the 2C TCR transgenic mouse model, in which the transgene is directed against H2d, it became possible to monitor the effector cells by staining with an antibody against the TCR transgene (1B2). By using this assay we showed that the marked deletion of 2C CD8 T cells by veto CTLs of DBA origin is markedly inhibited by MEK1/2 inhibitors such as U0126, reducing the veto activity from 86%±7% to 16%±14% in 6 experiments. In the present study we used anti pERK antibody to analyze by FACS the level of ERK phosphorylation in the effctor cells and we demonstrate that upon interaction of 2C effector cells (1B2+CD8+) with veto CTLs of H2d origin as opposed to CTLs of H2s origin, ERK1/2 are significantly more phosphorylated (21%±5.5% Vs.14%±2% phospho ERK1/2 cells out of the total 1B2+CD8+ cells, respectively). The upregulation of ERK1/2 phosphorylation in the 2C effector cells is associated with significant reduction of XIAP levels (36%±4% reduction at 24h and 54%±19% at 48h). This reduction of an apoptosis inhibitor which generally controls death receptor-induced apoptosis in lymphocytes is specifically induced by the veto cells of H2d origin recognized by the transegne TCR and not by non-relevant veto CTLs of H2s origin. Addition of U0126 MEK 1/2 inhibitor reverses this down regulation of XIAP (reduction of 15%±13% at 24h and only 7%±13% at 48h). Blockade of MHC-CD8 interaction with MHC class I α3-blocking Ab, or anti-CD8 (lyt 2.1) Ab partially inhibits the ERK1/2 phosphorylation (average inhibition in 4 experiments was 37%±13% and 28%±8%, respectively) and also prevents XIAP down regulation (18%±9% at 24h and 23%±13% at 48h). Considering that blockade by these antibodies generally leads to partial inhibition of the observed veto activity (44%±21% in 4 experiments), our present results showing partial inhibition of ERK phosphorylation and XIAP down regulation, are clearly relevant. Taken together, these results can explain in part why co-expression of CD8 and FasL on the veto CTLs is required for effective deletion of host anti-donor CD8 T cell clones. [Display omitted] Deletion of host anti-donor CD8 T cell clones by veto CTLs. CD8-MHC engagement induces pERK which is associated with reduction of XIAP levels, thereby enabling Fas-FasL apoptosis.

Clonal Expansion of Graft-Versus-Host Reactive CD8+ T Cells Is Dissociated from Full Effector Differentiation Following Delayed DLI.

Extrinsic factors within the host environment are crucial in determining recruitment of graft-versus-host (GVH)-reactive T cells to peripheral tissues and the capacity of these cells to induce graft-versus-host disease (GVHD). In this study, we have examined how the host environment influences graft-versus-leukemia (GVL) activity. Transfer of small numbers of allogeneic T cells to freshly irradiated (TBI allo) mice induces both GVL and GVHD, whereas transfer of much higher numbers of T cells to established (>8 weeks) mixed chimeras (MC) can induce GVL without GVHD. Using an EL4 tumor protection assay and low doses of B10.A splenocytes (3 × 10e6, a dose 1 log lower than normally transferred to MC to induce GVL), we observed that tumor-free survival of recipient mice was greater following transfer to TBI allo B6 recipients than in B6 + B10.A → B6 MC. To determine the mechanisms for this disparity, we used a BALB/c recipient/B6 donor strain combination, in which we could track the distribution of donor T cells within secondary lymphoid organs and bone marrow (BM) following transfer to TBI allo or MC recipients. Despite similar expansions within the spleen, there was a significant delay in the accumulation of polyclonal donor T cells (B6 CD45.1) and transgenic 2C GVH-reactive CD8+ T-cells (bearing TCR specific for recipient antigen) in the BM of MC compared to TBI allo mice. Moreover, in vivo cytotoxicity of host B cell targets occurred rapidly and was virtually complete in TBI allo recipients, but was absent in MC even at late time points. To evaluate the acquisition of effector functions in a clonal GVH-reactive CD8+ T cell population, we sorted 2C T cells at intervals following transfer and performed quantitative RT-PCR of molecules linked to effector differentiation. Strikingly, transcription of IFN-γ, granzyme B and TNF-α was significantly higher in cells derived from TBI allo hosts compared to MC. Similar differences in IFN-γ and granzyme B protein expression were confirmed in the polyclonal donor CD8+ T cell population. Since, full GVL activity might also depend upon the survival of GVH-reactive CTL, we also examined the viability of donor T cells during the initial response in both environments. We observed higher rates of sustained 2C CD8+ T cell apoptosis (as indicated by annexin V staining) following T cell transfer to MC. Furthermore, we also detected lower expression of common γ chain cytokine receptors that mediate responsiveness to IL-2, IL-7 and IL-15, upon donor T cells from MC. However, following secondary co-transfer to syngeneic recipients for 21 days, memory phenotype polyclonal donor CD8+ T cells derived from established MC (CD45.1+) were recovered to a greater extent than T cells initially derived from TBI allo mice (Thy1.1+), arguing against any intrinsic defect in the viability of GVH-reactive T cell populations emerging in the former setting. Indeed, when co-cultured in the presence of individual cytokines, MC-derived CD8+ T cells maintained viability to a greater (IL-2, IL-7) or equivalent (IL-15) extent as TBI allo-derived cells. Taken together, these data suggest that disparity in GVL activity following to TBI allo recipients and MC can be explained by differences in the effector function and survival of anti-host CTL in quiescent MC environment. Reduced GVL activity of donor T cells on a per-cell basis in MC can be compensated for by transferring greater numbers of cells.

Allopeptide‐Specific CD4+ T Cells Facilitate the Differentiation of Directly Alloreactive Graft‐Infiltrating CD8+ T Cells

To investigate the mechanism of CD4(+) T-cell help during the activation and differentiation of directly alloreactive CD8(+) T cells, we examined the development of obliterative airways disease (OAD) following transplantation of airways into fully mismatched recipient mice deficient in CD4(+) T cells. BALB/c trachea allografts became fibrosed significantly less frequently in B6 CD4(-/-) recipients as compared to wildtype controls. Furthermore, class I-directed cytotoxicity failed to develop in the absence of CD4(+) T cells. The infiltration of graft tissue by primed L(d)-specific directly alloreactive 2C CD8(+) T cells was not found to depend on the presence of CD4(+) T cells. Nevertheless, graft-infiltrating 2C CD8(+) T cells failed to express CD69 and granzyme B when CD4(+) T-cell help was unavailable. Importantly, reconstitution of B6 CD4(-/-) recipient mice with graft peptide-specific TCR-Tg CD4(+) T cells (OT-II or TEa) capable of recognizing antigen only on recipient APC allowed for full expression of CD69 and granzyme B by the directly alloreactive CD8(+) T cells and restored the capacity of recipients to reject their allografts. These results demonstrate that indirectly alloreactive CD4(+) T cells ensure the optimal activation and differentiation of graft-infiltrating directly alloreactive CD8(+) T cells independent of donor APC recognition.

The Immunologic Function of 1B2+ Double Negative (CD4−CD8−) T Cells in the 2C Transgenic Mouse 1

2C mice bearing the cytotoxic TCR for class I L(d) on a C57BL/6 (B6) background have a preponderance of 1B2+CD8+ T cells directed against L(d). These naive CD8+ T cells are not directly cytotoxic without prior in vivo or in vitro activation. However, after in vitro sensitization, they become highly cytotoxic and will acutely and specifically reject a tolerant L(d+) BALB/c heart graft. Anti-lymphocyte serum (ALS) treatment eliminates CD4+ and CD8+ cells and a large double negative (CD4-CD8-) 1B2+ non-cytotoxic transgenic cell population remains. The immunological function of this unique peripheral population of T cells is investigated in the 2C transgenic mouse. To determine the activation characteristics of the 2C CD4-CD8- T cells, 2C peripheral T cells were analyzed for 1B2+, CD8+, and CD4+ marker by FACS before and 48-h after 0.5 cc ALS i.p. Similarly, in vitro, the response of these 2C CD4-CD8- T cells remaining after deletion of mature CD4+ and CD8+ T cells with ALS plus complement were evaluated by mixed lymphocyte culture and cytotoxic T lymphocyte after 7 days culture with BALB/c, IL-2, or BALB/c + IL-2. Parallel experiments were performed with control non-transgenic B6 mice. Following in vitro culture with BALB/c + IL-2, 2C CD4-CD8- T cells were injected into B6 mice with a tolerant BALB/c heart (tolerization via anti-CD4 mAb and intrathymic BALB/c) to determine their immunogenicity. While peripheral T cells in control B6 mice have <5% CD4-CD8- cells, transgenic 2C mice have a significantly increased percentage at 29 to 35% (P < 0.01). After the deletion of CD4+ and CD8+ T cells with either in vivo or in vitro ALS, 2C CD4-CD8- T cells increased to 96 to 99%. After 7-day culture, the 2C CD4-CD8- T cells decreased again to 33 to 38%. Simultaneously, 2C CD8+ T cells decreased from 56 to 62% to 0.1 to 3% after ALS treatment, but again increased to 61 to 70% after in vitro culture. Untreated 2C cells responded to IL-2 or BALB/c antigen equally well. However, after ALS treatment, CD4-CD8- T cells responded to IL-2 and IL-2 plus antigen, but not BALB/c antigen alone. Finally, CD4-CD8- T cells cultured for 7 days with BALB/c + IL-2 rejected the tolerant BALB/c heart in 5.3 +/- 0.3 days. In the periphery of transgenic 2C mice is a unique CD4-CD8- population of T cells bearing the transgenic specific marker 1B2. These non-cytotoxic cells can be optimally stimulated to develop marked specific L(d) cytotoxicity in parallel with the expression of the CD8+ epitope.

Proliferation, Expansion, Effector Differentiation and Survival of GVH-Reactive T Cells Following Delayed DLI to Mixed Chimeras.

Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). DLI given immediately following lethal irradiation lead to severe GVHD. We examined the proliferation, expansion, differentiation and survival of GVH-reactive T cells following delayed DLI and compared the outcomes to those observed when identical DLI were administered early following lethal irradiation (TBI). MC recipients were prepared by TBI of BALB/c mice and reconstitution with mixed BALB/c and CD45.2 C57BL/6 (B6) T-cell depleted bone marrow (TCD BM). 10 weeks later, we transferred 1 x 107 CD45.1 B6 and 5 x 106 2C transgenic (tg) splenocytes. CD8+ T cells from 2C tg mice bear TCR specific for recipient class I MHC Ld. Polyclonal DLI-derived CD4+/CD8+ T cell responses were monitored by gating on CD45.1+ events and clonal 2C CD8+ T cell responses tracked using a clonotypic marker. For comparison, identical DLI together with TCD BM was administered to freshly irradiated BALB/c or B6 CD45.2 syngeneic recipients. MC recipients of delayed DLI developed a GVH reaction, as indicated by increases in donor chimerism, but no GVHD. In contrast, allogeneic recipients receiving DLI immediately following TBI developed lethal GVHD (median survival 32d vs. >100d post-delayed DLI, p<0.0001). By day 6, donor CD4+/CD8+ T cells had undergone almost equivalent proliferation as monitored by CFSE-dilution in MC and TBI allogeneic recipients. However, the kinetics and distribution of donor T cell expansion were distinct. Following delayed DLI, marked expansion of donor CD4+ cells (peak day 10) preceded expansion of CD8+ cells (peak day 13) in the spleen, with less accumulation in the lymph nodes, BM, liver and lung, and no accumulation in the gut. Histology revealed transient, mild lymphocytic infiltrates within the lung/liver but no evidence of colitis. In contrast, the kinetics of donor CD4+/CD8+ T cell expansion were more rapid in freshly irradiated recipients with CD4+/CD8+ responses peaking on day 4–7. The distribution was also different with major increases in donor CD4+/CD8+ numbers in the gut but less accumulation in the spleen. Histology confirmed severe colitis. The kinetics of proliferation, expansion and distribution of tg 2C CD8+ T cells showed a similar pattern to the polyclonal donor CD8+ T cell population. Following delayed DLI, 2C CD8+ T cells acquired a memory/activation phenotype (CD44hi, CD45RBlo, CD62Llo, CD49d+, CD27+) with similar kinetics to those observed in TBI mice developing GVHD. Despite the marked differences in trafficking to the gut, 2C CD8+ T cells in both groups of allogeneic recipients expressed equivalent levels of the gut homing receptor, α4β7. However, we also observed important differences: 1) IL-2Rαexpression was absent on 2C CD8+ T cells following delayed DLI, but was expressed at high levels (>50% by day +3) in freshly irradiated DLI recipients; 2) 2C CD8+ T cells showed greater reductions in expression of IL-7Rαfollowing delayed DLI; and 3) high rates of 2C CD8+ T cell apoptosis, as indicated by annexin V staining, were observed following delayed DLI with absolute numbers up to 6-fold greater than in TBI recipients. Thus, in contrast to responses in freshly irradiated mice that develop GVHD, GVH reactions induced by delayed DLI are characterized by delayed kinetics, a distinct distribution, marked apoptosis and reduced expression of cytokine receptors important for CD8+ T-cell survival.

Sequential Blockade and Engagement of Co-Stimulatory Pathways: A Potential Strategy for Amplifying Graft-Versus-Leukemia Responses without GVHD.

One potential approach for the effective disengagement of graft-versus-leukemia (GVL) effects from graft-versus-host disease (GVHD) following BMT is the use of nonmyeloablative conditioning as a platform for the adoptive transfer of donor T cells. In pre-clinical models, donor CD8+ T cells can induce powerful responses against tumors of host origin, but the effect lacks durability such that a re-challenge with tumor inevitability leads to tumor progression and death. This deficit is associated with the failure of functional CD8+ effector/memory T cells (TE/M) to survive long-term post-DLI. To examine the fate of GVH-reactive CD8+ T cells following DLI, we established mixed hematopoietic chimeras (MC) in a parent →F1 model using a nonmyeloablative protocol that incorporates co-stimulatory molecule blockade. B6D2F1 mice received 3Gy TBI and intra-peritoneal injections of anti-CD154 and anti-CD8 mAb on day 0 followed by infusion of 2 x 107 C57BL/6 bone marrow cells. 10 weeks later, when mAb had cleared from the circulation, MC received DLI that included CD8+ T cells from 2C transgenic mice that bear TCR specific for recipient class I MHC Ld. Using a clonotypic marker to monitor the response, we observed expansion of 2C CD8+ cells, peaking in the spleen on day 7 and then rapidly declining such that 2C CD8+ T cells were <0.1% of splenocytes by day 60. The decline in GVH-reactive T cells was associated with marked apoptosis and a sustained reduction in the expression of IL-7Rα. By day 60, no CTL activity against host cells was detectable. We reasoned that strategies that augment the survival of GVH TE/M might enhance the durability of the GVL response and, in the absence of tissue inflammation induced by conditioning, might not lead to GVHD. Co-stimulation through the tumor necrosis family receptor, OX40, which is expressed on activated T cells, is anti-apoptotic and enhances recruitment of TE/M to the memory pool. Following DLI, OX40 expression on 2C CD8+ T cells peaked on day 7 with somewhat earlier and sustained expression on DLI-derived CD4+ T cells. Since OX40 expression was specific for GVH-reactive T cells, we examined the effect of giving agonistic anti-OX40 antibody on day +5 following DLI. This was associated with rapid and complete conversion to full donor chimerism by day +14, whereas DLI + control antibody recipients had only partially converted by day +28. By day 60 post-DLI, anti-host CTL activity was clearly detectable in anti-OX40 recipients but not in controls. No clinical evidence of GVHD was observed, although histological examination revealed transient mild lymphocytic infiltration of the lamina propria on day +13, which resolved completely by day +18. In further experiments, anti-OX40 administration was associated with marked increases in the numbers of 2C CD8+ T cells in spleen, lymph node and bone marrow following DLI. Furthermore, effector differentiation, as assayed by intracellular expression of interferon-γ by 2C CD8+ T cells, was increased in recipients of anti-OX40 antibody. Of note, we observed a complete inhibition IL-7Rα down-regulation that is normally observed on activated CD8+ T cells following DLI. We conclude that OX40 co-stimulation following delayed DLI to established MC represents a potential means to enhance the magnitude and duration of a GVH reaction without the induction of significant GVHD.

The ICOS:ICOSL Costimulatory Pathway Plays an Important Role in GVHD and Bone Marrow (BM) Graft Rejection.

ICOS, a CD28/CTLA-4 family member, is expressed on activated T cells. ICOS Ligand, a B7 family member, is constitutively expressed on B cells, monocytes and some T cells. Through the use of blocking anti-ICOS mAb and ICOS deficient (−/−) mice, we found that ICOS:ICOSL interactions play an important role in GVHD and BM graft rejection. Anti-ICOS mAb (given d-1 to d28 post BMT) significantly delayed or reduced mortality at 2 different T cell doses in a full MHC-disparate GVHD model. ICOS−/− T cells led to delayed or reduced mortality at 3 different cell doses compared to wild-type T cells. ICOS−/− CD4+ or CD8+ T cells infused into class II- or class I-disparate recipients, respectively, revealed that ICOS:ICOSL interactions regulate both CD4+ and CD8+ T cell alloresponses. Anti-ICOS inhibited GVHD in a CD28-independent fashion. Anti-ICOS inhibited GVHD mediated by either stat 4−/− or stat 6−/− T cells indicating that the ICOS pathway regulates both Th2 and Th1-mediated GVHD. In contrast to blockade of the B7:CD28/CTLA-4, CD40L:CD40 or the OX40:OX40L pathway, anti-ICOS mAb inhibited GVHD even when delayed until d5 post BMT, a time when substantial T cell expansion has occurred. A TCR transgenic model of GVHD was used to further study effects of ICOS:ICOSL blockade. All CB6 F1 recipients of anti-host alloreactive 2C CD8+ and TEa CD4+ T cells succumbed to GVHD mortality by d18 after transfer of cells. In contrast, 88% of anti-ICOS-treated mice survived long-term. Evaluation of spleens early after transplant revealed that anti-ICOS mAb reduced the number of TEa CD4+ cells by 44% and 2C CD8+ cells by 83%. Green fluorescent protein (GFP) 2C CD8+ and GFP TEa CD4+ T cells were infused into irradiated CB6 F1 mice and irrelevant or anti-ICOS mAb was administered. Mice were imaged on d4, 7 and 12 after T cell transfer. By d7, pronounced infiltration of GFP+ cells was noted in the peripheral and mesenteric LN, spleen, Peyer's patches (PP), skin, gingiva, liver, kidney, lung, ileum, and colon of GVHD control mice. In contrast, there were fewer GFP+ cells in the spleen, ileum, colon, kidney, lung, skin and gingiva of anti-ICOS-treated mice, although there was no decrease in GFP+ cells in LNs or PP. To study the role of host ICOS expression in BM graft rejection, wild-type or ICOS−/− mice were sublethally irradiated and given allogeneic BM and evaluated for donor chimerism at 6 weeks post BMT. Five of 10 wild type mice engrafted (ave − 26% donor) in contrast to all 10 of ICOS−/− mice (ave − 71% donor). Collectively, these data indicate that ICOS:ICOSL interactions play an important role in GVHD, whether mediated by CD4+ Th1 or Th2 T cells or CD8+ T cells. Importantly, blockade of ICOS:ICOSL after initiation of alloresponses inhibited GVHD, in contrast to blockade of other costimulatory pathways, suggesting that the ICOS pathway may be a novel therapeutic target in primed transplantation situations. Anti-ICOS interfered with expansion of donor T cells in the spleen early after transplant and reduced the number of effector cells in several GVHD target tissues. These data suggest this pathway may be indicated for therapeutic targeting for the inhibition of GVHD and BM graft rejection.

Visualizing Allogeneic Bone Marrow (BM) Graft Rejection.

The process by which bone marrow is rejected by host T cells has not been able to be directly visualized to date. To study the process of allogeneic bone marrow rejection and the effects of therapeutic interventions, we created 2 models that allow us to 1) quantify the specific expansion of host-type alloreactive T cells early post-transplant in response to allogeneic BM infusion and to 2) image host T cells in vivo during BM rejection. For the first model, 2C and TEa lymph node (LN) cells were adoptively transferred into syngeneic C57BL/6 (B6) Rag deficient mice on d-2. 2C CD8+ and TEa CD4+ T cell receptor transgenic T cells are reactive against BALB/c alloantigen. Mice were irradiated with 200 cGy on d-1 and BALB/c BM was infused on d0. Controls included mice that received 2C/TEa LN cells but no BM. Ten days later, spleen analysis revealed that 2C CD8+ and TEa CD4+ T cells had expanded 322-fold and 33-fold (ave of 6 exp.), respectively, in mice receiving BALB/c BM compared to controls that did not receive BM. Expanded T cells were activated as determined by flow cytometric parameters and cell surface antigens. Data indicate that host alloreactive T cell expansion was inhibited by >95% by combined, but not single, costimulatory pathway blockade. Studies are in progress to analyze in vitro host anti-donor responses of adoptively transferred T cells. To visualize the response of host T cells to donor BM in vivo, we developed a rejection model for imaging involving the adoptive transfer of green fluorescent protein (GFP) T cells (obtained from GFP transgenic mice) into syngeneic non-GFP B6 recipients immediately following sublethal irradiation (500 cGy). Allogeneic BALB/c or syngeneic B6 BM was infused the following day. The syngeneic BMT controls allowed for the distinction of homeostatic vs alloreactive expansion of GFP+ T cells. Transplanted mice not receiving GFP T cells served as negative controls to verify lack of autofluorescence. Cohorts were imaged d4 to d18 post BMT. By d4, low numbers of GFP+ cells were evident in femoral BM cavity, peripheral and mesenteric LNs, spleen, Peyer's patches (PP) and to a lesser extent, lung. By d7, massive expansion of GFP+ cells could be visualized throughout the body of recipients of allogeneic BM. LNs, spleen, PP (peri-follicular area) and BM cavity increased dramatically in GFP intensity from d4 to d7. On d7 to d14, there were large foci of GFP+ cells in the lung, liver, skin, gingiva, kidney, uterus, and colon in allogeneic BMT recipients. Compared with allogeneic BMT recipients, syngeneic BMT recipients had greatly reduced numbers of GFP+ T cells in lymphoid organs and only rare cells were noted in liver, kidney, skin, BM and gingiva. In both allogeneic and syngeneic BMT recipients, lengths of ileum were diffusely infiltrated while other sections contained discrete foci of GFP+ cells. These imaging data provide a vivid illustration of the massive expansion and multi-organ distribution of host anti-donor T cells in vivo. Recent generation of GFP+ 2C and GFP+ TEa mice will permit the imaging of alloantigen-specific T cells during a rejection response. Additional imaging experiments are planned to study the fate of GFP+ BM transferred to allogeneic recipients under conditions of engraftment vs rejection. These models provide a unique platform for the testing of therapeutic interventions.

Trachea allograft class I molecules directly activate and retain CD8+ T cells that cause obliterative airways disease.

Human T cells responding against transplanted allogeneic lung tissue have been implicated in late graft failure secondary to obliterative bronchiolitis. This obliterative airways disease (OAD) also develops in heterotopic murine tracheal allografts in association with graft infiltration by both CD8(+) and CD4(+) T cells. To date, there has been little evidence to suggest that directly alloreactive CD8(+) T cells either promote chronic rejection or lead to the development of OAD following airway allotransplantation. Using L(d)-specific TCR-Tg 2C CD8(+) T cells adoptively transferred into wild-type B6 (H-2(b)) mice and the transplantation of BALB/c (H-2(d)) tracheal allografts, we now show that the direct recognition of donor-specific class I MHC molecules by host CD8(+) T cells leads to their activation, clonal expansion within the graft, and differentiation to an effector phenotype with the capacity to induce airway fibrosis. In addition, these experiments demonstrate that ongoing direct alloantigen recognition within the transplanted airway tissue is necessary for the recruitment and retention of these directly alloreactive CD8(+) T cells. Thus, these experiments are the first to definitively show a role for directly alloreactive CD8(+) T cells in the chronic rejection that leads to OAD.

Donor lymphocyte infusion-mediated graft-versus-leukemia effects in mixed chimeras established with a nonmyeloablative conditioning regimen: extinction of graft-versus-leukemia effects after conversion to full donor chimerism.

We investigated an approach to separating graft-versus-lymphoma (GVL) effects from graft-versus-host disease (GVHD) in mice receiving a nonmyeloablative conditioning regimen allowing establishment of mixed hematopoietic chimerism. We evaluated the ability of donor lymphocyte infusions (DLI) to mediate GVL effects without GVHD in mixed chimeras prepared with cyclophosphamide, anti-T-cell antibodies, and thymic irradiation. To examine the fate of GVH-reactive donor CD8+ T cells, we used the 2C T-cell receptor (TCR) transgenic mouse strain, which carries an Ld-specific transgenic TCR on the B6 background. Administration of DLI on day 35 post-BMT led to conversion from mixed to full donor chimerism and mediated a powerful GVL effect with complete protection (100% survival) against mortality induced by a host-type lymphoma (EL4) administered 7 days later (100% mortality in non-DLI controls; P<0.001). No GVHD occurred in DLI recipients. Rechallenging the surviving DLI recipients, which had converted to full chimerism, with the same tumor dose 17 weeks later led to rapid tumor mortality. Long-term DLI recipients had anti-host proliferative responses, but not CTL responses in vitro. When given as DLI together with wild-type spleen cells, marked expansion of GVH-reactive 2C CD8+ T cells was observed on day 10, followed by a marked decline in their numbers by week 10 post-DLI. Nonmyeloablative induction of mixed chimerism followed by administration of DLI can mediate powerful GVL effects. The late loss of DLI-mediated GVL effects may reflect the eventual loss of donor-derived GVH-reactive CTL, which occurs in association with conversion to full donor chimerism.