Search

Supplementary Figures 1-4. Supplementary Figure 1. In vitro suppressive function of tumor-infiltrating MDSC 3 days after LTI, and analysis of CD8+CD11+ dendritic cell tumor infiltration after LTI. Supplementary Figure 2. PDL-1 and CD62L expression of tumor-infiltrating cells. Supplementary Figure 3. CT26 luc+ tumors were established in BALB/c RAG2-/- animals. 30 Gy LTI was given on day 21 after tumor induction. Bioluminescence imaging (BLI) is shown with proton emission at serial time points. Supplementary Figure 4. Model for T cell mediated remissions and reversal of the immunosuppressive tumor microenvironment

Purpose: The goals of the study were to elucidate the immune mechanisms that contribute to desirable complete remissions of murine colon tumors treated with single radiation dose of 30 Gy. This dose is at the upper end of the ablative range used clinically to treat advanced or metastatic colorectal, liver, and non–small cell lung tumors.Experimental Design: Changes in the tumor immune microenvironment of single tumor nodules exposed to radiation were studied using 21-day (>1 cm in diameter) CT26 and MC38 colon tumors. These are well-characterized weakly immunogenic tumors.Results: We found that the high-dose radiation transformed the immunosuppressive tumor microenvironment resulting in an intense CD8+ T-cell tumor infiltrate, and a loss of myeloid-derived suppressor cells (MDSC). The change was dependent on antigen cross-presenting CD8+ dendritic cells, secretion of IFNγ, and CD4+T cells expressing CD40L. Antitumor CD8+ T cells entered tumors shortly after radiotherapy, reversed MDSC infiltration, and mediated durable remissions in an IFNγ-dependent manner. Interestingly, extended fractionated radiation regimen did not result in robust CD8+ T-cell infiltration.Conclusions: For immunologically sensitive tumors, these results indicate that remissions induced by a short course of high-dose radiotherapy depend on the development of antitumor immunity that is reflected by the nature and kinetics of changes induced in the tumor cell microenvironment. These results suggest that systematic examination of the tumor immune microenvironment may help in optimizing the radiation regimen used to treat tumors by adding a robust immune response. Clin Cancer Res; 21(16); 3727–39. ©2015 AACR.

Figure legends

Radiation therapy benefits the majority of patients across the spectrum of cancer types. However, both local and distant tumor recurrences limit its clinical success. While departing from the established tenet of fractionation in clinical radiotherapy, ablative-intensity hypo-fractionated radiotherapy, especially stereotactic radiosurgery (SRS) and stereotactic ablative radiotherapy (SABR), has emerged as an alternative paradigm achieving unprecedented rates of local tumor control. Direct tumor cell killing has been assumed to be the primary therapeutic mode of action of such ablative radiation. But with increasing recognition that tumor responses also depend on the immunostimulatory or immunosuppressive status of the tumor microenvironment, the immunologic effect of ablative radiotherapy is emerging as a key contributor to anti-tumor response. More recently, novel radiation modalities, such as spatially fractionated radiotherapy (SFRT) and ultra-high dose rate FLASH irradiation, that venture even further from conventional paradigms have shown promise of increasing the therapeutic index of radiation therapy with the potential of immunomodulation. Here, we review the immunomodulatory impact of novel radiation therapy paradigms, heretofore considered radiobiological heresies, a deeper understanding of which is imperative to realizing fully their potential for more curative cancer therapy.

Human invariant natural killer T (iNKT) cells are a rare innate-like lymphocyte population that recognizes glycolipids presented on CD1d. Studies in mice have shown that these cells are heterogeneous and are capable of enacting diverse functions, and the composition of iNKT cell subsets can alter disease outcomes. In contrast, far less is known about how heterogeneity in human iNKT cells relates to disease. To address this, we used a high-dimensional, data-driven approach to devise a framework for parsing human iNKT heterogeneity. Our data revealed novel and previously described iNKT cell phenotypes with distinct functions. In particular, we found 2 phenotypes of interest: (1) a population with T helper 1 function that was increased with iNKT activation characterized by HLA-II+CD161– expression, and (2) a population with enhanced cytotoxic function characterized by CD4–CD94+ expression. These populations correlate with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation and with new onset type 1 diabetes, respectively. Our study identifies human iNKT cell phenotypes associated with human disease that could aid in the development of biomarkers or therapeutics targeting iNKT cells.

Classical dendritic cells (cDCs) in mice have been divided into 2 major subsets based on the expression of nuclear transcription factors: a CD8+Irf8+Batf3 dependent (DC1) subset, and a CD8-Irf4+ (DC2) subset. We found that the CD8+DC1 subset can be further divided into CD8+DC1a and CD8+DC1b subsets by differences in surface receptors, gene expression, and function. Whereas all 3 DC subsets can act alone to induce potent Th1 cytokine responses to class I and II MHC restricted peptides derived from ovalbumin (OVA) by OT-I and OT-II transgenic T cells, only the DC1b subset could effectively present glycolipid antigens to natural killer T (NKT) cells. Vaccination with OVA protein pulsed DC1b and DC2 cells were more effective in reducing the growth of the B16-OVA melanoma as compared to pulsed DC1a cells in wild type mice. In conclusion, the Batf3-/- dependent DC1 cells can be further divided into two subsets with different immune functional profiles in vitro and in vivo.

Based on preclinical studies, combined kidney and hematopoietic cell transplantation was performed on fully HLA matched and haplotype matched patients at the Stanford University Medical Center. The object of the studies was to induce mixed chimerism, immune tolerance, and complete immunosuppressive drug withdrawal. Tolerance, persistent mixed chimerism, and complete withdrawal was achieved in the majority of fully matched patients. Persistent mixed chimerism and partial withdrawal has been achieved in the haplotype matched patients at present.

Radiation therapy, along with surgery and chemotherapy, is one of the main treatments for cancer. While radiotherapy is highly effective in the treatment of localized tumors, its main limitation is its toxicity to normal tissue. Previous preclinical studies have reported that ultra-high dose-rate (FLASH) irradiation results in reduced toxicity to normal tissues while controlling tumor growth to a similar extent relative to conventional-dose-rate (CONV) irradiation. To our knowledge this is the first report of a dose-response study in mice comparing the effect of FLASH irradiation vs. CONV irradiation on skin toxicity. We found that FLASH irradiation results in both a lower incidence and lower severity of skin ulceration than CONV irradiation 8 weeks after single-fraction hemithoracic irradiation at high doses (30 and 40 Gy). Survival was also higher after FLASH hemithoracic irradiation (median survival >180 days at doses of 30 and 40 Gy) compared to CONV irradiation (median survival 100 and 52 days at 30 and 40 Gy, respectively). No ulceration was observed at doses 20 Gy or below in either FLASH or CONV. These results suggest a shifting of the dose-response curve for radiation-induced skin ulceration to the right for FLASH, compared to CONV irradiation, suggesting the potential for an enhanced therapeutic index for radiation therapy of cancer.

Development of a new methodology to induce immunological chimerism after allogeneic hematopoietic cell (HC) transplantation in a rhesus macaque model is described. The chimeric state was achieved using a non-myeloablative, helical tomotherapy-based total lymphoid irradiation (TomoTLI) conditioning regimen followed by donor HC infusions between 1-haplotype matched donor/recipient pairs. The technique was tested as a feasibility study in an experimental group of seven rhesus macaques that received the novel TomoTLI tolerance protocol and HC allo-transplants. Two tomotherapy protocols were compared: TomoTLI (n = 5) and TomoTLI/total-body irradiation (TBI) (n = 2). Five of seven animals developed mixed chimerism. Three of five animals given the TomoTLI protocol generated transient mixed chimerism with no graft-versus-host disease (GVHD) with survival of 33, 152 and >180 days. However, the inclusion of belatacept in addition to a single fraction of TBI resulted in total chimerism and fatal GVHD in both animals, indicating an unacceptable conditioning regimen.

Replacement of failed organs followed by safe withdrawal of immunosuppressive drugs has long been the goal of organ transplantation. We studied changes in the balance of T cells and myeloid cells in the blood of HLA-matched and -mismatched patients given living donor kidney transplants followed by total lymphoid irradiation, anti-thymocyte globulin conditioning, and donor hematopoietic cell transplant to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In preclinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive polymorphonuclear (pmn) myeloid-derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of pmn MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes, including profound depletion of T cells and a marked increase in MDSCs in blood posttransplant. Posttransplant pmn MDSCs transiently increased expression of lectin-type oxidized LDL receptor-1, a marker of immunosuppression, and production of the T-cell inhibitor arginase-1. These posttransplant pmn MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous T-cell receptor microbead-stimulated pretransplant T cells when cocultured in vitro. In conclusion, we elucidated changes in receptors and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to those of MDSCs required for tolerance in mice. These trials were registered at www.clinicaltrials.gov as #NCT00319657 and #NCT01165762.

The International Workshop on Clinical Transplant Tolerance is a biennial meeting that aims to provide an update on the progress of studies of immunosuppression minimization or withdrawal in solid organ transplantation. The Fourth International Workshop on Clinical Tolerance was held in Pittsburgh, Pennsylvania, September 5-6, 2019. This report is a summary of presentations on the status of clinical trials designed to minimize or withdraw immunosuppressive drugs in kidney, liver, and lung transplantation without subsequent evidence of rejection. All protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. The workshop also included presentations of mechanistic studies designed to improve understanding of the cellular and molecular basis of tolerance and to identify potential predictors/biomarkers of tolerance. Strategies to enhance the safety of hematopoietic cell transplantation and to improve patient selection/risk stratification for clinical trials were also discussed.

Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)–matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype–matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.

The resurgence of haploidentical stem cell transplantation (HaploSCT) over the last decade is one of the most important advances in the field of hematopoietic stem cell transplantation (HSCT). The modified platforms of T cell depletion either ex vivo (CD34+ cell selection, "megadoses" of purified CD34+ cells, or selective depletion of T cells) or newer platforms of in vivo depletion of T cells, with either post-transplantation high-dose cyclophosphamide or intensified immune suppression, have contributed to better outcomes, with survival similar to that in HLA-matched donor transplantation. Further efforts are underway to control viral reactivation using modified T cells, improve immunologic reconstitution, and decrease the relapse rate post-transplantation using donor-derived cellular therapy products, such as genetically modified donor lymphocytes and natural killer cells. Improvements in treatment-related mortality have allowed the extension of haploidentical donor transplants to patients with hemoglobinopathies, such as thalassemia and sickle cell disease, and the possible development of platforms for immunotherapy in solid tumors. Moreover, combining HSCT from a related donor with solid organ transplantation could allow early tapering of immunosuppression in recipients of solid organ transplants and hopefully prevent organ rejection in this setting. This symposium summarizes some of the most important recent advances in HaploSCT and provides a glimpse in the future of fast growing field.

Murine models showed that CD8+CD44hi memory T (TM) cells could eradicate malignant cells without inducing graft-versus-host disease (GVHD). We evaluated the feasibility and safety of infusing freshly isolated and purified donor-derived phenotypic CD8+ TM cells into adults with disease relapse after allogeneic hematopoietic cell transplantation (HCT). Phenotypic CD8 TM cells were isolated after unmobilized donor apheresis using a tandem immunomagnetic selection strategy of CD45RA depletion followed by CD8+ enrichment. Fifteen patients received CD8+ TM cells at escalating doses (1 × 106, 5 × 106, or 10 × 106 cells per kg). Thirteen received cytoreduction before CD8+ TM cell infusion, and 9 had active disease at the time of infusion. Mean yield and purity of the CD8+ TM infusion were 38.1% and 92.8%, respectively; >90% had CD8+ T effector memory phenotype, cytokine expression, and secretion profile. No adverse infusional events or dose-limiting toxicities occurred; GVHD developed in 1 patient (grade 2 liver). Ten patients (67%) maintained or achieved response (7 complete response, 1 partial response, 2 stable disease) for at least 3 months after infusion; 4 of the responders had active disease at the time of infusion. With a median follow-up from infusion of 328 days (range, 118-1328 days), median event-free survival and overall survival were 4.9 months (95% confidence interval [CI], 1-19.3 months) and 19.6 months (95% CI, 5.6 months to not reached), respectively. Collection and enrichment of phenotypic CD8+ TM cells is feasible, well tolerated, and associated with a low incidence of GVHD when administered as a manipulated infusion of donor lymphocytes in patients who have relapsed after HCT. This trial was registered at www.clinicaltrials.gov as #NCT01523223.

Cancer therapy has developed around the concept of killing, or stopping the growth of, the cancer cells. Molecularly targeted therapy is the modern expression of this paradigm. Increasingly, however, the realization that the cancer has co-opted the normal cells of the stroma for its own survival has led to the concept that the tumor microenvironment (TME) could be targeted for effective therapy. In this review, we outline the importance of tumor-associated macrophages (TAM), a major component of the TME, in the response of tumors to cancer therapy. We discuss the normal role of macrophages in wound healing, the major phenotypes of TAMs, and their role in blunting the efficacy of cancer treatment by radiation and anticancer drugs, both by promoting tumor angiogenesis and by suppressing antitumor immunity. Finally, we review the many preclinical studies that have shown that the response of tumors to irradiation and anticancer drugs can be improved, sometimes markedly so, by depleting TAMs from tumors or by suppressing their polarization from an M1 to an M2 phenotype. The data clearly support the validity of clinical testing of combining targeting TAMs with conventional therapy. Clin Cancer Res; 23(13); 3241–50. ©2017 AACR.

The combination of total lymphoid irradiation and anti-T-cell antibodies safely induces immune tolerance to combined hematopoietic cell and organ allografts in humans. Our mouse model required host natural killer T (NKT) cells to induce tolerance. Because NKT cells normally depend on signals from CD8+ dendritic cells (DCs) for their activation, we used the mouse model to test the hypothesis that, after lymphoid irradiation, host CD8+ DCs play a requisite role in tolerance induction through interactions with NKT cells. Selective deficiency of either CD8+ DCs or NKT cells abrogated chimerism and organ graft acceptance. After radiation, the CD8+ DCs increased expression of surface molecules required for NKT and apoptotic cell interactions and developed suppressive immune functions, including production of indoleamine 2,3-deoxygenase. Injection of naive mice with apoptotic spleen cells generated by irradiation led to DC changes similar to those induced by lymphoid radiation, suggesting that apoptotic body ingestion by CD8+ DCs initiates tolerance induction. Tolerogenic CD8+ DCs induced the development of tolerogenic NKT cells with a marked T helper 2 cell bias that, in turn, regulated the differentiation of the DCs and suppressed rejection of the transplants. Thus, reciprocal interactions between CD8+ DCs and invariant NKT cells are required for tolerance induction in this system that was translated into a successful clinical protocol.

Nonmyeloablative total lymphoid irradiation and antithymocyte globulin (TLI-ATG) conditioning is protective against graft-versus-host disease (GVHD), while retaining graft-versus-tumor activity across various hematologic malignancies. We report our comprehensive experience using TLI-ATG conditioning in 612 patients with hematologic malignancies who underwent allogeneic transplantation at Stanford University from 2001 to 2016. All patients received granulocyte colony-stimulating factor–mobilized peripheral blood grafts and cyclosporine and mycophenolate mofetil for GVHD prophylaxis. The median age was 60 years (range, 21-78), with a median follow-up of 6.0 years (range, 1.0-16.4). Common diagnoses included acute myeloid leukemia (AML; n = 193), myelodysplastic syndrome (MDS; n = 94), chronic lymphocytic leukemia (CLL; n = 80), non-Hodgkin lymphoma (NHL; n = 175), and Hodgkin lymphoma (HL; n = 35). Thirty-four percent of patients had a comorbidity index ≥3, 30% had a high to very high disease risk index, and 56% received unrelated donor grafts, including 15% with HLA-mismatched donors. Ninety-eight percent underwent transplant in the outpatient setting, and 57% were never hospitalized from days 0 through 100. The 1-year rates of nonrelapse mortality (NRM), grade II-IV acute GVHD, and extensive chronic GVHD were 9%, 14%, and 22%, respectively. The 4-year estimates for overall and progression-free survival were 42% and 32% for AML, 30% and 21% for MDS, 67% and 43% for CLL, 68% and 45% for NHL, and 78% and 49% for HL. Mixed chimerism correlated with the risk of relapse. TLI-ATG conditioning was well tolerated, with low rates of GVHD and NRM. Durable remissions were observed across hematologic malignancies, with particularly favorable outcomes for heavily pretreated lymphomas. Several efforts are underway to augment donor chimerism and reduce relapse rates while maintaining the favorable safety and tolerability profile of this regimen.

The goals of tolerance in patients with solid organ transplants are to eliminate the lifelong need for immunosuppressive (IS) drugs and to prevent graft loss due to rejection or drug toxicity. Tolerance with complete withdrawal of IS drugs has been achieved in recipients of HLA-matched and mismatched living donor kidney transplants in 3 medical centers using hematopoietic cell transplants to establish mixed or complete chimerism.

Conventional local tumor irradiation (LTI), delivered in small daily doses over several weeks, is used clinically as a palliative, rather than curative, treatment for chemotherapy-resistant diffuse large B-cell lymphoma (DLBCL) for patients who are ineligible for hematopoietic cell transplantation. Our goal was to test the hypothesis that accelerated, but not conventional, LTI would be more curative by inducing T cell–mediated durable remissions. We irradiated subcutaneous A20 and BL3750 lymphoma tumors in mice with a clinically relevant total radiation dose of 30 Gy LTI, delivered in 10 doses of 3 Gy over 4 days (accelerated irradiation) or as 10 doses of 3 Gy over 12 days (conventional irradiation). Compared with conventional LTI, accelerated LTI resulted in more complete and durable tumor remissions. The majority of these mice were resistant to rechallenge with lymphoma cells, demonstrating the induction of memory antitumor immunity. The increased efficacy of accelerated LTI correlated with higher levels of tumor cell necrosis vs apoptosis and expression of “immunogenic cell death” markers, including calreticulin, heat shock protein 70 (Hsp70), and Hsp90. Accelerated LTI–induced remissions were not seen in immunodeficient Rag-2−/− mice, CD8+ T-cell–depleted mice, or Batf-3−/− mice lacking CD8α+ and CD103+ dendritic cells. Accelerated, but not conventional, LTI in immunocompetent hosts induced marked increases in tumor-infiltrating CD4+ and CD8+ T cells and MHCII+CD103+CD11c+ dendritic cells and corresponding reductions in exhausted PD-1+Eomes+CD8+ T cells and CD4+CD25+FOXP3+ regulatory T cells. These findings raise the possibility that accelerated LTI can provide effective immune control of human DLBCL.

The Third International Workshop on Clinical Tolerance was held in Stanford, California, September 8-9, 2017. This is a summary of Workshop presentations of clinical trials designed to withdraw or minimize immunosuppressive (IS) drugs in kidney and liver transplant patients without subsequent evidence of rejection. All clinical protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. Tolerance to HLA matched and mismatched living donor kidney transplants with complete withdrawal of IS drugs without subsequent rejection for up to 14 years of observation was achieved in more than 50 patients enrolled in trials in four medical centers after the establishment of transient or persistent chimerism. Complete IS drug withdrawal without chimerism was reported in a prospective trial of liver transplantation combined with injection of regulatory T cells. IS drug minimization without rejection was reported in recipients of living donor kidney transplants enrolled in the One Study consortium after injection of recipient regulatory T cells, or injection of donor regulatory monocytes or dendritic cells. In conclusion, considerable progress has been made in achieving IS drug withdrawal after cell therapy in recipients of organ transplants.

Current theory holds that macrochimerism is essential to the development of transplant tolerance. Hematopoietic cell transplantation from the solid organ donor is necessary to achieve macrochimerism. Over the last 10–20 years, trials of tolerance induction with combined kidney and hematopoietic cell transplantation have moved from the preclinical to the clinical arena. The achievement of macrochimerism in the clinical setting is challenging, and potentially toxic due to the conditioning regimen necessary to hematopoietic cell transplantation and due to the risk of graft-versus-host disease. There are differences in chimerism goals and methods of the three major clinical stage tolerance induction strategies in both HLA-matched and HLA-mismatched living donor kidney transplantation, with consequent differences in efficacy and safety. The Stanford protocol has proven efficacious in the induction of tolerance in HLA-matched kidney transplantation, allowing cessation of immunosuppressive drug therapy in 80% of study participants, with the safety profile of conventional transplantation. In HLA-mismatched transplantation, multi-lineage macrochimerism of over a year’s duration can now be consistently achieved with the Stanford protocol, with complete withdrawal of immunosuppressive drug therapy during the second post-transplant year as the next experimental step and test of tolerance.

Allogeneic hematopoietic cell transplantation after failed autologous transplant for lymphoma using TLI and anti-thymocyte globulin conditioning

Many patients lack a fully HLA-matched donor for hematopoietic cell transplantation (HCT), and HLA mismatch is typically associated with inferior outcomes. Total lymphoid irradiation and antithymocyte globulin (TLI-ATG) is a nonmyeloablative conditioning regimen that is protective against graft-versus-host disease (GVHD), and we hypothesized that the protective effect would extend beyond HLA-matched donors. We report outcomes for all consecutively transplanted patients at Stanford University from December 2001 through May 2015 who received TLI-ATG conditioning and HCTs from 8 to 9 out of 10 HLA-mismatched unrelated donors (MMUDs, N = 72) compared with 10 out of 10 HLA-matched unrelated donors (MUDs, N = 193). The median age of the patients was 60 years with a median follow-up of 2 years, and there was a similar distribution of lymphoid and myeloid malignancies in both cohorts. There were no significant differences between MMUD and MUD cohorts in overall survival (46% vs 46% at 5 years, P = .86), disease-free survival (38% vs 28% at 5 years, P = .25), nonrelapse mortality (17% vs 12% at 2 years, P = .34), acute GVHD grades III-IV (6% vs 3% at day +100, P = .61), or chronic GVHD (39% vs 35% at 5 years, P = .49). There was a trend toward less relapse in the MMUD cohort (45% vs 60% at 5 years, hazard ratio: 0.71, P = .094), which was significant for patients with lymphoid malignancies (29% vs 57% at 5 years, hazard ratio: 0.55, P = .044). Achieving full donor chimerism was strongly associated with lower relapse rates. TLI-ATG conditioning may overcome the traditionally poorer outcome associated with HLA-mismatched donors and may be particularly well suited for patients with lymphoid malignancies who lack HLA-matched donors.

IgG autoantibodies, including antibodies to double-stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus (SLE), but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG in vitro. Despite the rarity of iNKT cells in lupus blood (0.002-0.05% of CD3-positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti-dsDNA IgG by lupus PBMCs. Addition of anti-iNKT or anti-CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B-cell maturation. Like fresh iNKT cells, expanded iNKT-cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti-dsDNA. This activity was inhibited by anti-CD40L antibody, as well as anti-CD1d antibody, confirming a role for CD40L-CD40 and TCR-CD1d interactions in lupus iNKT-cell-mediated help. These results reveal a critical role for iNKT cells in B-cell maturation and autoantibody production in patients with lupus.

Human invariant natural killer T cells (iNKT) are a rare population of lymphocytes that bridges innate and adaptive immune functions. These cells show diverse phenotypes and function, but the current population structure imposed on iNKT cells inadequately differentiates these diverse phenotypes and function. Using single-cell RNA-sequencing and subsequent functional assays and flow cytometric analysis, we present a novel population structure that characterizes human iNKT cell heterogeneity. Markers delineating primary iNKT cells for IL-4+, IFNg+TNFa+, and cytotoxicity functions were CD4, KLRG1, and CD94, respectively. In addition, iNKT cells follow a temporal evolution of function and associated markers - upon chronic activation, HLA-DR expression associated with increased Th1 skewing. Unsupervised clustering of our iNKT single-cell RNA-sequencing data from a graft-versus-host disease (GvHD) and a GvHD-free post-transplant patient showed a GvHD-enriched subset that is defined by expression of HLA-DR alleles. In a cohort of 38 patients, 19 of whom developed GvHD, flow cytometric analysis of iNKT cells revealed increased HLA-DR and decreased CD161 expression when compared to GvHD-free patients. CD94 expression was overall increased after allogeneic hematopoietic cell transplantation. Functional assessment of HLA-DRhiCD161− iNKT cells showed that this population produced significantly less IL-4 while maintaining IFNg production. Furthermore, high expression of HLA-DR could be detected before initial GvHD onset compared to in patients who did not develop GvHD. Together, our data suggests HLA-DR as a marker for delineating iNKT cells with a proinflammatory phenotype and is associated with GvHD. In sum, our approach for describing iNKT cell population structure revealed new markers that associate with different iNKT cell phenotypes. This new paradigm for describing iNKT cell heterogeneity can potentially be translated into the clinic as an immune monitoring tool for patients undergoing allogeneic hematopoietic cell transplantation.

Tolerance to combined kidney and hematopoietic cell transplant has been achieved in humans after establishment of mixed chimerism allowing for the withdrawal of immunosuppressive drugs. The seminal contributions of Ray Owen provided the scientific basis for the human protocol.

Sixteen patients conditioned with total lymphoid irradiation and anti-thymocyte globulin were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T cell subsets, and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft versus host disease (GVHD), and 8 with chimerism for at least 6 months were withdrawn from anti-rejection medications for 1 to 3 years (mean- 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and 4 patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naïve CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, total lymphoid irradiation and anti-thymocyte globulin promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

We used a model of combined bone marrow and heart transplantation, in which tolerance and stable chimerism is induced after conditioning with fractionated irradiation of the lymphoid tissues and anti–T-cell antibodies. Graft acceptance and chimerism required host CD4+CD25+ Treg production of IL-10 that was in-turn enhanced by host invariant natural killer (NK) T-cell production of IL-4. Up-regulation of PD-1 on host Tregs, CD4+CD25− conventional T (Tcon) cells, and CD8+ T cells was also enhanced by NKT cell production of IL-4. Up-regulated PD-1 expression on Tregs was linked to IL-10 secretion, on CD8+ T cells was linked to Tim-3 expression, and on CD4+ Tcon cells was associated with reduced IFNγ secretion. Changes in the expression of PD-1 were induced by the conditioning regimen, and declined after bone marrow transplantation. In conclusion, NKT cells in this model promoted changes in expression of negative costimulatory receptors and anti-inflammatory cytokines by Tregs and other T-cell subsets in an IL-4–dependent manner that resulted in tolerance to the bone marrow and organ grafts.

Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation markedly changes the balance of residual T cell subsets to favor CD4+CD44hi NKT cells because of the differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results showed that CD4+ T cells were markedly more resistant than CD8+ T cells, and CD44hi T cells, including NKT cells and memory T cells, were markedly more resistant than CD44lo (naive) T cells. The memory T cells immunized to alloantigens persisted even after myeloablative (1000 cGy) TBI and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1000 cGy was prevented in p53−/− mice, there was progressive T cell death in p53−/− mice at higher doses. Although p53-dependent T cell death changed the balance of subsets, p53-independent T cell death did not. In conclusion, resistance of CD44hi T cells to p53-dependent cell death results in the persistence of immunological memory after TBI and can explain the immune-mediated rejection of marrow transplants in sensitized recipients.

Background: Nonmyeloablative total lymphoid irradiation and antithymocyte globulin (TLI-ATG) conditioning is associated with a low risk of graft-versus-host disease (GVHD) and non-relapse mortality (NRM) while retaining graft-versus-tumor activity across a range of lymphoid and myeloid malignancies. Prior studies of TLI-ATG conditioning have been limited by the relatively small sample size and short duration of follow up. Herein we report mature data from a large, single-institution cohort of patients with lymphoid and myeloid malignancies who underwent allogeneic transplantation using TLI-ATG conditioning. Methods: Patients: We included all consecutively transplanted patients at Stanford University Hospital from December 2001 through December 2016 who underwent allogeneic hematopoietic cell transplantation (HCT) using TLI-ATG conditioning for lymphoid or myeloid malignancies. All patients received G-CSF-mobilized peripheral blood hematopoietic cells and cyclosporine and mycophenolate mofetil for GVHD prophylaxis. Statistical analysis: The data were analyzed as of December 31, 2017, allowing for a minimum follow up of 1 year for all patients. For time-to-event analyses, the Kaplan-Meier method was used to estimate overall survival (OS), progression-free survival (PFS), and the cumulative incidence of acute and chronic GVHD and NRM. An exploratory analysis was done to identify factors associated with mortality and relapse. Log-rank tests were used to calculate differences between groups. Results: Patient characteristics: 613 patients underwent allogeneic HCT with TLI-ATG conditioning at a median age of 60 years (range 21-78 years) with a median follow up of 5.1 years (range 1.0-15.3 years). Diagnoses included acute myeloid leukemia (AML, N=194), myelodysplastic syndrome (MDS, N=93), myelofibrosis (N=10), chronic myeloid leukemia (N=12), acute lymphoblastic leukemia (N=11), chronic lymphocytic leukemia (CLL, N=83), non-Hodgkin lymphoma (NHL, N=175), and Hodgkin lymphoma (HL, N=35). 44% of patients had HLA-matched related, 41% had HLA-matched unrelated, and 15% had HLA-mismatched unrelated donors. 34% of patients had a comorbidity index ≥3, and 34% of patients had a disease risk index (DRI) of high or very high. 55% of lymphoma patients had failure of prior autologous HCT. Safety and tolerability: Patient outcomes are shown in Figure 1. Among all 613 patients, the cumulative incidences of acute GVHD grade 2-4 and grade 3-4 at day +100 were 11% and 4%, respectively, and the cumulative incidence of extensive chronic GVHD was 21% at 1 year. NRM was 7% at 1 year, and was consistently low across high-risk subgroups including older adults ≥65 years old (9% at 1 year), patients with a comorbidity index ≥3 (9% at 1 year), and patients lacking an HLA-matched related donor (10% at 1 year). 49% of patients remained outpatient without hospitalization during the first 100 days post-transplant. Long-term outcomes: For the five most common diagnoses, the 4-year OS and PFS estimates were 41% and 32% for AML, 31% and 23% for MDS, 66% and 42% for CLL, 67% and 46% for NHL, and 79% and 46% for HL, respectively. Factors associated with mortality included a high or very high DRI (HR 2.11, 95% CI 1.65-2.69, p Conclusions: TLI-ATG conditioning was well tolerated with a low risk of GVHD and NRM even among high-risk subgroups, including septuagenarians, patients with multiple comorbidities, and patients lacking an HLA-matched related donor. Nearly half of all patients remained outpatient during the first 100 days post-transplant. Long-term OS and PFS were seen across a range of hematologic malignancies, with particularly favorable outcomes for lymphoma patients, most of whom had failure of prior autologous HCT. Mixed chimerism correlates with the risk of relapse, and efforts are underway to augment donor chimerism and reduce relapse rates. Figure 1. Figure 1. Disclosures Miklos: Kite - Gilead: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Genentech: Research Funding. Muffly:Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding.

Human invariant natural killer T cells (iNKT) are a rare population of lymphocytes that bridges innate and adaptive immune functions. iNKT cells have different potent effector functions and subsets, but this heterogeneity is mostly understood by a superimposed framework of classic T cell markers. This may help explain contradictory studies of iNKT cells in diseases for which they are thought to play a pathogenic role. In this study, we present data suggesting new markers to understand human iNKT cell heterogeneity and function, as well as monitoring this in the clinical context of allogeneic stem cell transplantation (ASCT) and graft-versus-host disease (GVHD). In both preclinical murine models and in correlative clinical studies, iNKT cells are associated with less GVHD and better immune reconstitution following ASCT. We performed bulk whole transcriptome sequencing on iNKT cells from patients at day +30 after ASCT, some who developed and some who did not develop GVHD. Gene expression signatures of the iNKT cells from each individual are grouped based on a transcriptome library of reference cell types (CIBERSORT). Using this approach, we distinguished patients who will develop GVHD as predominately having an activated cytotoxic cell gene signature, whereas patients without GVHD had a CD4+ T cell gene signature within purified peripheral blood iNKT cells. Using two different high throughput single-cell RNA sequencing (sc-seq) platforms to determine differential gene expression on the single cell level, we first examined the gene expression signatures of primary human iNKT cells from healthy donors. Activation with CD3/28 microbeads of primary iNKT cells promoted two main differential transcriptional profiles in iNKT cells. One profile resembles conventional CD4+ T cells and associated Th2 cytokine profile (IL2, IL4), whereas the other profile has genes associated with cytotoxicity and inflammation (TNF, IFNG, GZMB). Confirmation by flow cytometry showed that while CD8 surface expression did not well differentiate the pro-inflammatory and cytotoxic subsets, but both CD94 and KLRG1 better associate with Th1 and cytotoxic responses. CD94+ iNKT are restricted to CD4- cells and KLRG1 expressed in both CD4+ and CD4- populations. By comparison, the analysis of sc-seq of normal healthy subjects and post-transplant patients with and without GVHD resulted in the identification of three major iNKT populations. The population most associated with GVHD showed expression of a pro-inflammatory profile enriched for CXCR4,CD94, HLA-DRB1/A1 and decreased KLRB1. Whereas, post-transplant patients with healthy GVHD-free and relapse free immune reconstitution showed a more T cell like profile but with increased expression of CXCR4, CD94, KLRB1 and decreased HLA-DRB1/A1. Using flow cytometry of ex vivo expanded iNKT cells, we found the expansion of CXCR4+HLA-DR+ that can be CD4+ or CD4- and that these cells produce high levels of the cytokine TNF-a, IFN-g and IL-4. Importantly, iNKT cells that are CXC4+HLA-DR+KLRB1low make significantly less IL-4 and these are enriched in the GVHD setting. Using flow cytometry, we measured iNKT cell phenotypes in a pilot cohort of 48 individuals; 10 healthy controls, 10 patients with no complications or relapse after ASCT and 18 patients with GVHD of which 9 were steroid refractory. In addition, we evaluated 11 patients with new onset T1D. The frequency of the cytotoxic CD94+ iNKT cells were significantly increased in all patients following and in T1D compared to healthy controls. Whereas, patients with GVHD showed an increase in HLA-DR+CXCR4+KLRB1low iNKT cells. Increases in the late activation marker HLA-DR distinguished corticosteroid refractory (HLA-DR++) from steroid responsive patients at the time of initial GVHD diagnosis. For patients who responded to steroids or further therapy, we found that a reduction in HLA-DR associated with treatment response. In a second 24 patient cohort, we found that the presence HLA-DR+CXCR4+CD161low cells at day +30 was statistically greater in patients who would go on to develop GVHD as compared as those who did not (student T test, p To conclude, we have used different methods to discover and validate groups of iNKT cells with distinct functions, and the associated phenotype correlate with clinical inflammation both following ASCT and in T1D. Disclosures No relevant conflicts of interest to declare.

Vaccination with tumor Ags has not been an effective treatment for solid tumors. The goal of the current study was to determine whether a combination of vaccination and hematopoietic cell transplantation (HCT) can effectively treat primary, disseminated, or metastatic CT26 and MC38 murine colon tumors. Vaccination of tumor-bearing mice with irradiated tumor cells and CpG adjuvant failed to alter progressive tumor growth. However, mice bearing primary, disseminated lung, or metastatic liver tumors were uniformly cured after administration of total body irradiation, followed by the transplantation of hematopoietic progenitor cells and T cells from syngeneic, but not allogeneic vaccinated donors. Requirements for effective treatment of tumors included irradiation of hosts, vaccination of donors with both tumor cells and CpG, transfer of both CD4+ and CD8+ T cells along with progenitor cells, and ability of donor cells to produce IFN-γ. Irradiation markedly increased the infiltration of donor T cells into the tumors, and the combined irradiation and HCT altered the balance of tumor-infiltrating cells to favor CD8+ effector memory T cells as compared with CD4+CD25+FoxP3+ T regulatory cells. The combination of vaccination and autologous hematopoietic cell transplantation was also effective in treating tumors. In conclusion, these findings show that otherwise ineffective vaccination to solid nonhematologic tumors can be dramatically enhanced by HCT.

Although it is well known that in vivo radiation depletes immune cells via the Bcl-2 apoptotic pathway, a more nuanced analysis of the changes in the balance of immune-cell subsets is needed to understand the impact of radiation on immune function. We show the balance of T-cell subsets changes after increasing single doses of total body irradiation (TBI) or after fractionated irradiation of the lymphoid tissues (TLI) of mice due to differences in radioresistance and Bcl-2 expression of the NKT-cell and non-NKT subsets to favor CD4+Bcl-2hi NKT cells. Reduction of the Bcl-2lo mature T-cell subsets was at least 100-fold greater than that of the Bcl-2hi subsets. CD4+ NKT cells upregulated Bcl-2 after TBI and TLI and developed a Th2 bias after TLI, whereas non-NKT cells failed to do so. Our previous studies showed TLI protects against graft versus host disease in wild-type, but not in NKT-cell-deficient mice. The present study shows that NKT cells have a protective function even after TBI, and these cells are tenfold more abundant after an equal dose of TLI. In conclusion, differential expression of Bcl-2 contributes to the changes in T-cell subsets and immune function after irradiation.

We describe a recipient of combined kidney and hematopoietic-cell transplants from an HLA-matched donor. A post-transplantation conditioning regimen of total lymphoid irradiation and antithymocyte globulin allowed engraftment of the donor's hematopoietic cells. The patient had persistent mixed chimerism, and the function of the kidney allograft has been normal for more than 28 months since discontinuation of all immunosuppressive drugs. Adverse events requiring hospitalization were limited to a 2-day episode of fever with neutropenia. The patient has had neither rejection episodes nor clinical manifestations of graft-versus-host disease.

Lupus-prone NZB/W F1 mice develop glomerulonephritis after T helper cell-dependent isotype switching of autoantibody secretion from IgM to IgG at about 6 months of age. We compared innate immune natural killer (NK) T cells and conventional T cells for their capacity to help spontaneous in vitro immunoglobulin and autoantibody secretion of innate immune (B-1 and marginal zone) and conventional (follicular) B cell subsets from NZB/W F1 mice. We found that purified NKT cells not only increased spontaneous secretion of IgM and IgM anti-double-stranded (ds)DNA antibodies by B-1 and marginal zone B cells, but also facilitated secretion of IgG anti-dsDNA antibodies predominantly by B-1 B cells. Few IgM or IgG anti-dsDNA antibodies were secreted by follicular B cells, and conventional T cells failed to provide potent helper activity to any B cell subset. All combinations of T and B cell subsets from normal C57BL/6 mice failed to generate vigorous IgM and IgG secretion. NZB/W NKT cell helper activity was blocked by anti-CD1 and anti-CD40L mAb. In conclusion, direct interactions between innate immune T and B cells form a pathway for the development of IgM and IgG lupus autoantibody secretion in NZB/W mice.

Background. Posttransplant total lymphoid irradiation (TLI) treatment has been applied to tolerance induction protocols in heart and kidney transplantation models. Methods. We examined the efficacy and mechanism of posttransplant TLI treatment in the induction and maintenance of tolerance in a rat orthotopic liver transplantation model. Results. Posttransplant TLI prolonged ACI (RT1 a ) liver allograft survival in Lewis (RT1 b ) hosts, with 50% long-term engraftment without immunosuppression and without evidence of chronic rejection. Injection of donor-type liver mononuclear cells (LMCs) facilitated the prolongation of graft survival, with more than 70% of grafts in LMC recipients surviving more than 100 days without chronic rejection. Recipients with long-term liver allograft survival accepted ACI but not PVG skin grafts. In TLI-conditioned recipients with accepted grafts, apoptosis occurred predominantly in graft-infiltrating leukocytes. In contrast, there were few apoptotic leukocytes in rejecting grafts. Recipients with long-term graft acceptance (>100 days of survival) demonstrated evidence of immune deviation; mixed lymphocyte reaction to ACI stimulator cells was vigorous, but secretion of interferon-γ and interleukin-2 was reduced. In tolerant recipients, the number of Foxp3 + CD25 + CD4 + regulatory T cells was increased in the liver allograft as well as in the peripheral blood. Conclusion. We conclude that posttransplant TLI induces tolerance to liver allografts via a mechanism involving apoptotic cell-deletion and immunoregulation.

Purpose: The goals of the study were to elucidate the immune mechanisms that contribute to desirable complete remissions of murine colon tumors treated with single radiation dose of 30 Gy. This dose is at the upper end of the ablative range used clinically to treat advanced or metastatic colorectal, liver, and non–small cell lung tumors. Experimental Design: Changes in the tumor immune microenvironment of single tumor nodules exposed to radiation were studied using 21-day (>1 cm in diameter) CT26 and MC38 colon tumors. These are well-characterized weakly immunogenic tumors. Results: We found that the high-dose radiation transformed the immunosuppressive tumor microenvironment resulting in an intense CD8+ T-cell tumor infiltrate, and a loss of myeloid-derived suppressor cells (MDSC). The change was dependent on antigen cross-presenting CD8+ dendritic cells, secretion of IFNγ, and CD4+T cells expressing CD40L. Antitumor CD8+ T cells entered tumors shortly after radiotherapy, reversed MDSC infiltration, and mediated durable remissions in an IFNγ-dependent manner. Interestingly, extended fractionated radiation regimen did not result in robust CD8+ T-cell infiltration. Conclusions: For immunologically sensitive tumors, these results indicate that remissions induced by a short course of high-dose radiotherapy depend on the development of antitumor immunity that is reflected by the nature and kinetics of changes induced in the tumor cell microenvironment. These results suggest that systematic examination of the tumor immune microenvironment may help in optimizing the radiation regimen used to treat tumors by adding a robust immune response. Clin Cancer Res; 21(16); 3727–39. ©2015 AACR.

Keywords: health services and outcomes research; clinical research / practice; kidney transplantation / nephrology; economics; tolerance; tolerance: clinical

Chronic graft-vs-host disease (GVHD) is a major cause of morbidity and mortality of long-term survivors of allogeneic hemato-poietic cell transplantation (HCT). Chronic GVHD can have features of an autoimmune collagen vascular disease with clinical manifestations similar to autoimmune scleroderma and systemic lupus erythematosus (SLE). However, the pathogenesis of chronic GVHD is poorly understood. It is unclear how autoreactive T and B cells are generated in chronic GVHD recipients. We have recently developed a new chronic GVHD model by transplantation of donor DBA/2 (H-2d) spleen cells into major histocompatibility complex (MHC)-matched but minor antigen-mismatched sublethally irradiated BALB/c (H-2d) recipients as well as athymic BALB/cnu/nu and adult-thymectomized BALB/c recipients. Both euthymic and athymic BALB/c recipients developed high levels of serum IgG autoantibodies, sclerodermatous skin damage, and glomerulonephritis. Disease induction required both donor CD25-CD4+ T and B cells in transplants. In contrast, donor CD25+CD4+ T regulatory (Treg) cells prevented the disease induction. These results indicate that host thymus is not required for induction of chronic GVHD and that quiescent autoreactive T and B cells in transplants from nonautoimmune donors may be activated and expanded to cause chronic GVHD with autoimmune manifestations in allogeneic recipients, and donor Treg cells can suppress this process.

We identified committed T cell progenitors (CTPs) in the mouse bone marrow that have not rearranged the TCRβ gene; express a variety of genes associated with commitment to the T cell lineage, including GATA-3, T cell-specific factor-1, Cβ, and Id2; and show a surface marker pattern (CD44+CD25−CD24+CD5−) that is similar to the earliest T cell progenitors in the thymus. More mature committed intermediate progenitors in the marrow have rearranged the TCR gene loci, express Vα and Vβ genes as well as CD3ε, but do not express surface TCR or CD3 receptors. CTPs, but not progenitors from the thymus, reconstituted the αβ T cells in the lymphoid tissues of athymic nu/nu mice. These reconstituted T cells vigorously secreted IFN-γ after stimulation in vitro, and protected the mice against lethal infection with murine CMV. In conclusion, CTPs in wild-type bone marrow can generate functional T cells via an extrathymic pathway in athymic nu/nu mice.