Homeostatic Peripheral Expansion Research Articles

PB CD8 T N and CD4 T EM on day30 after Allo-HSCT Is a Predictive Marker for the Development of aGVHD

Introduction ： Acute GVHD (aGVHD) is a common complication 20-60 days after allo-HSCT and can be life-threatening in some cases. It has been reported to be associated with T-cell immune reconstitution, but the detailed mechanisms are not clear. Early post-transplant lymphocyte recovery is mainly due to homeostatic peripheral expansion (HPE), which is variable depending on various factors such as donor source, recipient's age. T cell-related cytokines, such as sIL-2R , IL-7 and TNFr1 are known biomarkers for the aGVHD, but there are few reports that predict the aGVHD incidence rate using the peripheral blood (PB) lymphocyte subset. We evaluated the effect of PB lymphocyte subset in early post-transplant period on aGVHD. Methods ： We retrospectively analyzed 83 patients (40 in AML, 12 in ALL, 1 in MPAL, 13 in MDS, 2 in MPN, 9 in malignant lymphoma/LPD and 3 in AA) who underwent the allo-HSCT in our hospital from 2013 to 2023. The median age of patients was 44 years (range:17-66). Sixteen received BM, 47 received PB (including 28 with haploidentical (9 with ATG, 19 with PTCY)), and 20 received CB. GVHD prophylaxis was FK506+MTX in 61 patients, CyA+MTX in 3 and MMF+FK506+CY in 19. The median observation period was 495 [38-2983] days. PB lymphocyte subset (Naïve Tcell (T N):CD45RA+CD62L+, Central memory Tcell (T CM) :CD45RA-CD62L+, Effector memory Tcell (T EM) :CD45RA-CD62L-, Effector memory Tcell re-expressed CD45RA (T EMRA) :CD45RA+CD62L-, Th1:CD3+4+CXCR3+CCR6-, Th2:CD3+4+CXCR3-CCR6-, Th17:CD3+4+CXCR3-CCR6+) at day30 (range:28-35) after transplantation were analyzed by FCM. Results ： Twenty-five patients had grade II-IV aGVHD, and 11 had grade Ⅲ-IV aGVHD. Patients with Grade II-IV aGVHD exhibited significantly lower proportion of CD8+T N as compared to non aGVHD groups (GVHD+: 17.7 ± 13.3%; GVHD-: 30.9 ± 16.3%; p < 0.001) . In contrast, the proportion of CD4+T EM and CD8+T EM were higher in the Grade II-IV acute GVHD group (GVHD+: 30.9 ± 23.1%; GVHD-: 21.9 ± 15.6%; p=0.04, GVHD+: 36.1 ± 16.2%; GVHD-: 22.7 ± 14.7%; p < 0.001, respectively). We found a strong statistical correlation between the proportion of CD8+T N and CD8+T EM. The cumulative incidences of Grade II-IV and III -IV acute GVHD at day + 100 for CD8+T N <20% group was 56.9% and 29.6% compared with 14.2% and 6.3% for the CD8+T N >20% group (p < 0.001 and p < 0.01). Similarly, the cumulative incidences of Grade II-IV and III -IV acute GVHD at day + 100 for CD4+T EM >30% group was 48.1% and 32.6% compared with 22.2% and 5.9% for the CD4+T EM <30% group (p= 0.02 and p < 0.01). Two-year NRM was higher in the CD8+T N <20% group than in the CD8+T N >20% group (26.3% vs 4.2%, p=0.008). The two-year cumulative incidence of relapse (CIR) was comparable between the two groups, but tended to be lower in the mismatched CR group (CD8+T N <20%: 33.1% versus CD8+T N >20%: 43.4%, p=0.31). Conclusion ： PB CD8 T N and CD4 T EM fraction on day30 is a predictive biomarker for the development of acute GVHD.

Cytokine Release from Stimulated PBMC after-HSCT: Results of a Post-Transplant Zinc Oral Supplementation Trial (ZENITH)

Introduction: the immune system may take months to recover after hematopoietic stem cell transplantation (HSCT). One important factor involved in the immune reconstitution is the recovery of the thymic production of a new T-cell repertoire, the other is the homeostatic peripheral expansion of mature lymhpocytes (HPE). It is known that HPE affect in a negative way the killing functions of the mature lymphocytes and their ability in secreting cytokines. On the other hand, the production of recent thymic emigrants (RTEs) by the thymus confers a broader spectrum against pathogens, but it needs a longer time. Zinc is fundamental for immune cells: several studies demonstrated that Zinc causes regrowth of thymic epithelial cells (TECs) in adults, improving the production of RTEs. Many studies showed the ability of Zinc to improve the in vitro cytokine release from immune cells, and others demonstrated its anti-apoptotic role on mature lymphocytes. As described elsewhere, we performed a clinical trial with the aim of investigating a potential therapeutic role of Zinc after HSCT (ZENITH trial). Here we illustrate the results of the cytokine release assays performed on the peripheral blood mononucleated cells (PBMC) isolated from the patients participating to the ZENITH trial.Patients and methods: From January 2015 to May 2016, we prospectively enrolled 19 patients affected by multiple myeloma (12 males, 7 females) undergoing single MEL 100 or 200 auto-SCT after one or two lines of therapy and stem cell collection. The trial, prospective and randomized, has been approved by local ethics committee (EUDRACT: 2014-004499-47). All patients undersigned the informed consent. The clinical trial was carried out in accordance with Helsinki declaration. Randomization was performed at day 0 (day of PBSC infusion). Ten patients were treated from day +5 to day +100 after transplant only with standard antimicrobial prophylaxis, whereas nine patients received in addition 600 mg/day of Zinc sulfate (uncoated tablets). Peripheral blood samples were collected in both groups at 4 timepoints (T0: day of stem cell collection; T1: day before conditioning; T2: day +30 after HSCT; T3: day +100 after HSCT). Absolute lymphocyte count was performed with an automatic counter. PBMC were obtained by separation on Ficoll gradient. 3x105/well cells were seeded in 48 w's plates and stimulated with RPMI 5%AS, PHA 2µg/ml. Supernatants were collected after both 3 and 5 days from stimulation and immediately frozen. Cytokines in the supernatants were evaluated by sandwich ELISA using R&D System Duo Set assays. All the values are expressed in picograms/ml. We dosed the levels of 4 cytokines: gamma-IFN, IL-2, IL-5 and alpha-TNF. On the basis of the literature, we considered the day +3 as the best time to measure IL-5 and TNF and the day +5 for IFN and IL-2. Non-parametric tests have been used for case-control analyses and for intra-group diachronic analyses. Spearman test has been performed for correlation tests.Results: The median levels of IFN increased both in the Zinc and in the Control group at day +100 after transplant. The release of IL-2 was higher in the Zinc group at T0; then, it was reduced respect to pre-transplant level at T2 in both groups, whereas it slightly rose from T2 until T3. The secretion of IL-5 in the Control group decreased from T0 until T3, whereas it increased from T2 until T3 in the Zinc group. TNF levels in T1 were higher than in T0 in both groups. Their levels remained stable at T2, but their median value increased in the Zinc group at T3, when they were reduced in the Control group (p=0.046). All the other above-described trends did not reach statistically significance, due to the still low number of patients enrolled. There was no correlation between absolute lymphocyte count and cytokine levels (p>0.05).Conclusion: preliminary data from our pilot study show that the secretion of different cytokines undergoes changes before and after HSCT. Moreover, PBMC isolated from patients treated with Zinc supplementation presented higher levels of IL-2, IL-5 and TNF at day +100 after HSCT when stimulated with PHA. The ability of releasing cytokine depends from the functional status of lymphocytes and the most represented T-cells subpopulations. Studies with larger cohorts of patients and stimulation test on separated T-lymphocyte populations will clarify whether the Zinc may contribute in the rejuvenation of immune system after transplant. [Display omitted] DisclosuresGalimberti:Novartis: Speakers Bureau; Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Incyte: Speakers Bureau.

NLRP12 protein function in the regulation of MHC-I expression in monocytes of patients transplanted with HPCs and their relationship to the reconstitution of CD8 T lymphocytes. (P1333)

Abstract The reconstitution of the T CD8 lymphocyte population in patients subject to hematopoietic cell transplantation (HCT) depends largely of homeostatic peripheral expansion (HPE) of the T CD8 lymphocytes that were not eliminated during the pre-transplant conditioning regimen or the ones that are contained in the transplanted cells. In order to have an efficient HPE, MHC-I molecules, IL-7 and IL-15 are required. Evidences obtained in our lab indicate that the low and/or heterogeneous MHC-I levels of expression correlate with a inefficient reconstitution of T CD8 lymphocytes in patients that received allogeneic HCT. The deficiency in MHC-I expression could be due to a decrease in the expression of the NLRP12 protein, which has been proposed as a positive regulator of classical and non-classical MHC-I molecules expression. Therefore the objective of the following project is to determine the involvement of NALP12 in the MHC-I expression in monocytes from patients subject to HCT. We studied a cohort of 11 patients, 6 of which received autologous transplant and 5 with allogeneic transplant. Most of the patients present normal levels of T CD8 lymphocytes and a homogeneous expression of MHC-I. The patients that received autologous transplantation show a correlation of r=0.56. Our results suggest that NALP12 could act as an enhancer for the expression of the MHC-I molecules and that this expression could be affected in patients subject to allogeneic HPC transplantation.

A two-hit model of autoimmunity: lymphopenia and unresponsiveness to TGF-β signaling

A two-hit model of autoimmunity: lymphopenia and unresponsiveness to TGF-β signaling

W5-2 Lymphopenia-induced proliferation (LIP) triggers the development of follicular helper T (T<sub>FH</sub>) cells which have a distinctive ontogeny and a critical role in aberrant B cell responses

Lymphopenia results in the homeostatic peripheral expansion of lymphocytes to maintain T cell homeostasis. Accumulating data show that lymphopenia-induced proliferation (LIP) has a pathogenic role in the development of autoimmunity in animal models and human systemic autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome and rheumatoid arthritis. To clarify the mechanism of anti-nuclear antibodies (ANA) production, we established a lymphopenic mouse model in which CD4+ T cell subsets from wild-type BALB/c mice were adoptively injected into BALB/c nude mice, which induced IgG-type ANA production. We observed that class switching and ANA production were enhanced when regulatory T cells were depleted. We identified IL-21-producing PD-1+ TFH cells which develop from conventional T cells during LIP and drive germinal center reactions with aberrant B cell responses. Compared with traditional TFH cells, this subset has a distinctive ontogeny and a critical role in breaking B cell tolerance. This study reveals the physiological mechanism how immunological tolerance is maintained during LIP and would help to understand the pathogenesis of systemic autoimmune diseases.

Exploitation of differential homeostatic proliferation of T-cell subsets following chemotherapy to enhance the efficacy of vaccine-mediated antitumor responses

The 5-year survival rate for stage IB-III non-small-cell lung cancer (NSCLC) remains 15%. Surgical resection followed by adjuvant chemotherapy with cisplatin and vinorelbine is one standard-of-care. We sought to determine in a preclinical model whether (a) the combination of cisplatin and vinorelbine could positively modulate components of the immune system independent of antitumor activity, and (b) there were synergistic effects of this drug combination and vaccine immunotherapy. We examined the effect of cisplatin/vinorelbine on gene expression, cell-surface phenotype, and CTL-mediated cytolysis of murine lung carcinoma cells in vitro; we also assessed the effects of cisplatin/vinorelbine on immune subsets and function of Tregs in vivo. Finally, we evaluated the potential synergy between chemotherapy and a recombinant yeast-CEA vaccine in a murine model transgenic for CEA with mice bearing lung tumors. These studies demonstrate that exposure of lung tumor cells to the platinum doublet cisplatin/vinorelbine modulates tumor cell phenotype and increases sensitivity to CTL-mediated cytolysis. These studies also demonstrate that cisplatin/vinorelbine (a) induces sub-myeloablative leucopenia that differentially modulates reconstitution of Treg versus effector T-cell subsets and (b) can be employed synergistically with vaccine, exploiting homeostatic peripheral expansion of T cells. Antitumor studies show for the first time that cisplatin/vinorelbine combined with vaccine increases the survival of mice with established NSCLC. These findings provide the rationale for the potential clinical benefit of the combined use of vaccine with cisplatin/vinorelbine chemotherapy regimens.

Gvhd Increases Apoptosis of CD8+, but Not CD4+, T Cells Expanded by Vaccines but Is Not Dependent on Fas Ligand

AbstractAbstract 2098Donor lymphocyte infusions (DLIs) administered following allogeneic blood and marrow transplantation (alloBMT) can enhance T cell reconstitution through homeostatic peripheral expansion (HPE), but can also cause graft-versus-host-disease (GVHD). Vaccines are a strategy to skew T cells toward a “third-party” tumor antigen, but it is not clear if the effects of GVHD on vaccine-responding T cells is equivalent to the effects on alloantigen-reactive polyclonal T cells. Previous work by others has demonstrated “bystander” effects of GVHD causing activation-induced cell death through the Fas pathway on polyclonal T cells; however, the impact of GVHD on T cells expanded by a vaccine expressing a third-party nonalloantigen remains unclear. T cell depleted (TCD) minor histocompatibility antigen (mHA)-mismatched alloBMT (CD45.1+ B6 –> CD45.1+/CD45.2+ B6 × C3H.SW) was followed by a CD45.1+ B6 DLI on day +14 to induce GVHD. CD45.2+ B6 Rag2-/- T cell receptor transgenic (TCRTg) CD4+ or CD8+ T cells specific for epitopes derived from the male histocompatibility antigen complex (HY) were administered on day +28 with an HY-expressing male dendritic cell vaccine to determine the impact of the allogeneic environment on the persistence, proliferation and survival of vaccine-responding T cells. Despite the fact that syngeneic and allogeneic recipients had similar degrees of lymphopenia, the absolute number of CD4+ and CD8+ TCRTg T cells was decreased 5 and 7 days respectively after adoptive transfer and vaccination in alloBMT recipients compared to syngeneic recipients (p < 0.05). To determine the mechanism of decreased persistence of the TCRTg T cells, proliferation was measured by assessing dilution of CFSE and apoptosis was measured by FACS analysis for Annexin V. Importantly, since both the CD4+ and CD8+ TCRTg T cells do not undergo HPE, all proliferation has to be antigen-driven. Neither CD4+ nor CD8+ TCRTg T cells proliferated in the absence of HY vaccine following alloBMT, confirming lack of bystander proliferation. Interestingly, both CD4+ (p < 0.01) and CD8+ (p < 0.05) TCRTg T cells undergo less vaccine-induced proliferation after alloBMT compared to syngeneic recipients. The amount of CD8+ TCRTg T cell apoptosis following vaccination was higher in allogeneic recipients (p < 0.05), but there was no difference in CD4+ TCRTg T cell apoptosis. Surprisingly, using a DLI from Fas ligand-deficient B6 (gld) donors on day +14 was sufficient to induce GVHD but did not prevent CD8+ TCRTg T cell apoptosis. Thus, diminished vaccine responses during GVHD appear to result in part from impaired CD8+ and CD4+ T cell vaccine-driven proliferation, but there is an additional contribution on CD8+ T cell apoptosis that is not dependent on recognition of alloantigen or the Fas-Fas ligand pathway. Disclosures:No relevant conflicts of interest to declare.

Immune Reactions Following Reduced-Intensity Allogeneic Stem Cell Transplantation (Allo-CST): Role of Plasma IL-7 and IL-15 Levels, and Their Receptors

AbstractAbstract 1251Allogeneic stem cell transplantation (allo-SCT) following reduced-intensity conditioning (RIC) represents a concrete alternative to standard myeloablative conditioning. Even though, immune reactions observed after RIC (i.e. GVHD, GVL.), tend to be similar to those observed after myeloablative regimens, there are many differences in their clinical features, timing of onset, and their impact on patient's outcome. The homeostatic cytokines, IL-7 and IL-15 are essential driving forces for homeostatic peripheral expansion of T lymphocytes that are responsible, not only for GVL effect but also for GVHD which is a major complication post-transplant. Almost all mature T-cells express the specific IL-7 receptor alpha chain (IL-7Rα). Memory CD8+ T cells and natural killer (NK) cells express the highest levels of IL-2/15Rβ chain, making these cells particularly responsive to IL-15.Few studies have associated GVHD with systemic levels of either IL-7 or IL-15 but only one concerned patients undergoing RIC. However, patients reported in the latter study, had received prior induction chemotherapy and experienced profound lymphopenia after conditioning, leading to levels of IL-7 and IL-15 comparable to those usually observed after myeloablative regimens. In addition, the issue of IL-7R and IL-2/15Rβ expression has not been addressed yet in allo-SCT patients. This prompted us to prospectively investigate plasma levels of IL-7 and IL-15, T-cell recovery, and the expression levels of IL-7Rα and IL-2/15Rβ chains in patients undergoing RIC.Forty-five consecutive patients who underwent fully HLA-matched allo-SCT after RIC were included. Plasma levels of IL-7 and IL-15 were determined by ELISA at enrolment, on day 0 before grafting, every three days during the first month, and then on days 60 and 90. CD3+, CD4+, CD8+ T-cells and NK cells counts at day 30, 60 and 90 post-graft were obtained by flow-cytometry-based technique. Expression of IL-7Rα and IL-2/15Rβ (% and MFI) was evaluated on each subsets of naïve and memory T-cells, categorized according to their expression of CD45RA and CCR7 markers.Kinetic courses of plasma IL-7 levels, evolved inversely to lymphocyte counts up to day 30 (R= -.529; P= .001). The higher plasma IL-7 levels were associated with the lower MFI of IL-7Rα on CD4+ naïve, CD8+ naïve, CD4+ central memory, and CD8+ central memory T-cells (p=.005, .01, <.001 and .005, respectively). IL-15 levels peaked on day +7 to rapidly return to a normal range. No statistically significant correlation was found between IL-15 levels and the expression of IL-2/15Rβ. A positive correlation was evidenced between IL-15 and CRP levels on day +7 (R= .488; P = .013) and day +14 (R= .621; P< .001) while the correlation between IL-7 and CRP levels at the same time points reached marginal statistical significance.A total of 16 (35%) recipients developed grades 2–4 acute GVHD (aGVHD) at a median time of 42 days post graft. By day +30, IL-7 and IL-15 levels were slightly correlated with aGVHD grades (R=.30; P=.05 and R=.31; P = .048, respectively) and inversely correlated with the time to onset (IL-7: R= -.334; P= .031 and IL-15: R= -.367; P= .018). In those patients, there was a diminished surface densities of IL-7Rα chain in both the naïve (P= .029) and the central memory (P= .043) CD4+ T-cells. In multivariate analysis, IL-7 level measured on day +30 was the foremost predictive factor for grade 2–4 aGVHD (P= .002).Collectively, this study indicates that the level of IL-7 depends rather on the level of lymphopenia whereas IL-15 is more sensitive to the inflammatory context. A high level of IL-7 induces a downregulation of IL-7Rα on naïve and central memory CD4+ T cells, already known to contain alloreactive T cells. The determination of plasma level of IL-7 by day+30 would help predict aGVHD. This predictive time is less precocious than those observed after myeloablative conditioning in agreement with the later development of aGVHD observed with RIC regimens. Disclosures:No relevant conflicts of interest to declare.

Impact of Donor IL-7Rα Polymorphism On Recipient Plasma IL-7 levels and Acute Gvhd Following Allogeneic Stem Cell Transplantation.

AbstractAbstract 1464IL-7 is one of essential driving forces for homeostatic peripheral expansion of T lymphocytes that are responsible, not only for GVL effects but also for acute GVHD, a major post-transplant complication. High plasma levels of IL-7 in the early phase post-transplant, has been associated with high incidence of severe acute GVHD regardless the intensity of conditioning regimen. Inter-individual variations have also been reported. Here we aimed to identify factors that could have an impact on IL-7 level and, therefore, on acute GVHD. This prompted us to prospectively investigate plasma levels of IL-7, T-cell subsets recovery, T cells' IL-7Rα chain expression, and IL-7Rα chain polymorphism in 100 pts who underwent fully HLA-matched allogeneic stem cell transplantation in our unit. Pts received either myeloablative (n= 60) or nonmyeloablative (n=40). Forty donors were unrelated. Source of stem cells, was bone marrow in 71 pts and PBCS in 29. Sex ratio (M/F) was (66/34) and median age at transplant was of 49 years.Plasma IL-7 level was determined by ELISA at enrolment, on day 0 before grafting, every three days during the first month, and then on days 60 and 90.CD3+, CD4+, CD8+ T-cells and NK cells counts at day 30, 60 and 90 post-graft were obtained by flow-cytometry-based technique. Expression of IL-7Rα (% and MFI) was evaluated on each subset of naïve and memory T-cells, categorized according to their expression of CD45RA and CCR7 markers.The detection of IL-7Ra single nucleotide polymorphism (SNPs) by sequence specific PCR (SSP), in donors, was carried out as described by Shamim et al, (BMT 2006). IL-7 receptor consisted of γc-chain and specific α-chain. A range of IL7R α-chain SNPs was reported (+510 C/T, +1237 A/G, +2087 T/C which all resulted in amino-acid substitution).At the time of analysis, 40 (40%) recipients had developed grade 2–4 acute GVHD (aGVHD) with a median time of 33 days post transplant.As expected, IL-7 levels peaked around the second week at median of 11.5 pg/mL (0.4-30.2) after transplant. Kinetic courses of plasma IL-7 levels, evolved inversely to lymphocyte counts up to d+30 (p<.001). The cumulative incidence of aGVHD was higher if by day+18 pts had IL-7 levels above the median concentration (p= .046). A higher level of IL-7 at day+18 was confirmed as a predictive factor of subsequent risk of aGVHD (HR= 1,079; 95% CI: 1.022 – 1.139; p= .006). By calculating the area under the curve of IL-7 between d-15 and d+30, we observe that a high exposure to IL-7 during the first month is correlated with the risk of aGVHD (p=.002).IL-7 plasma levels were inversely correlated with IL-7Rα expression only on central/effector memory CD4+ and central/effector memory CD8+, and terminally differentiated CD8+ T-cells (p =.006, .013, .044, .001 and .028, respectively). Of note, at d+30, pts had 85% (34-99) and 86% (23-99) of CD4+ and CD8+ memory T cells, respectively.Contrary to +1237 A/G and +2087 T/C, donor's +510 CC or CT was the only polymorphism to be associated with higher level of plasma IL-7 in recipients during the first month post-transplant in particular at d+18, predictive date for aGVHD (p = .026). In multivariate analysis, pts who received graft from donor with +510CC or CT experienced more often grade 2–4 aGVHD than those with +510 TT (P = .049).Collectively, this study confirms the role of IL-7 in grade 2–4 aGVHD. Indeed, the high level of IL-7 that down regulates IL-7Rα, could suggest activation and consumption of IL-7 by alloreactive T cells, including those involved in aGVHD development. By difference in affinity and cytokine consumption, the polymorphism +510 of donor t-cell IL-7R α-chain might explain, in part, the wide variation of IL-7 level among pts. Disclosures:No relevant conflicts of interest to declare.

Homeostatic Expansion and Phenotypic Conversion of Human T Cells Depend on Peripheral Interactions with APCs

Immune recovery in lymphopenic hosts depends largely on homeostatic peripheral expansion, especially when thymopoiesis is insufficient, as is often the case in human adults. Although it has been well studied in mice, the study of homeostatic peripheral expansion of human T cells has been limited by the lack of an appropriate in vivo model. In this study, we use T cell-deficient humanized mice and an adoptive transfer approach to demonstrate that two distinct proliferative responses of autologous T cells occur in vivo in a lymphopenic setting. Human naive CD4 and CD8 T cells that undergo rapid proliferation acquire a memory-like phenotype and the ability to rapidly produce IFN-gamma, whereas those undergoing slow proliferation retain naive phenotypic and functional characteristics. Recovery of both populations depends on the extent of human non-T cell chimerism in the periphery of recipient humanized mice. Furthermore, memory conversion of CD4 and CD8 T cells correlates with the level of human CD14+ and CD19+ chimerism in recipient mice, respectively, suggesting that different types of APCs support memory conversion of CD4 and CD8 T cells. Because lymphopenia affects clinical outcomes, this model, which will allow detailed investigation of the effects of lymphopenia in patients, is of clinical significance.

Plasma levels of IL-7 and IL-15 in the first month after myeloablative BMT are predictive biomarkers of both acute GVHD and relapse

T-cell reconstitution after allo-SCT initially depends on homeostatic peripheral expansion of donor T cells, the level of which may promote the differentiation of alloreactive and tumor-reactive effectors. IL-7 and IL-15 exert their effect as key homeostatic cytokines. We prospectively investigated plasma levels of IL-7 and IL-15 in a homogeneous group of 40 patients in CR of their hematologic malignancy undergoing myeloablative, fully (10/10) HLA-matched BMT. IL-7 and IL-15 proceeded along similar kinetic courses, peaking at wide ranges (3.8-30.2 and 14.3-66 pg/ml, respectively) on day +14 when all patients were profoundly lymphopenic. Occurrence and grade of subsequent acute GVHD were significantly associated with heightened day +14 IL-7 and IL-15 levels. Association of peak IL-7 level to grade 2-4 acute GVHD was confirmed by Cox multivariate analysis (hazard ratio (HR)=5.38; P=0.022). Malignancy relapse was significantly associated with reduced day +14 levels of IL-15 (Cox multivariate analysis: HR=0.93; P=0.035). Plasma IL-7 and IL-15 levels in the early post transplantation period are therefore biomarkers that can help predict subsequent development of acute GVHD and malignancy relapse.

Regulatory T cells in the control of T cell homeostasis

Lymphopenia results in homeostatic peripheral expansion of lymphocytes in order to maintain T-cell homeostasis. T-cell homeostasis requires the regulation of lymphocytes numbers, function, and diversity responsive to foreign antigen and the maintenance of self-tolerance during lymphopenia-induced proliferation. Accumulating data elucidate that regulatory T cells critically contribute to the control of T cell homeostasis. Moreover, recent studies show that regulatory T cell homeostasis requires the co-existence with conventional T cells which can produce IL-2. Thus, T-cell homeostasis is maintained by the close interactions between regulatory T cells and conventional T cells. Understanding the mechanisms of homeostatic proliferation will lead to new therapeutic interventions for autoimmune diseases.

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

Low MHC-I density impairs the homeostatic proliferation of CD8 T cells after HSCT

We hypothesise that the modifications in the Major Histocompatibility Class I (MHC-I) expression after Hematopoietic Stem Cell Transplantation (HSCT) might impact on CD8 T cell recovery. To evaluate our proposal, we measured the number of peripheral CD8 T cells and the MHC-I expression on the monocytes from the patients who underwent allogenic or autologous HSCT. Three months after transplantation, almost all of the autologous recipients showed a high density of MHC-I and the CD8 T cell counts were within the normal range. In contrast, allogeneic recipients had low or heterogeneous expression of MHC-I and low numbers of CD8 T cells. Similar patterns were observed even after 6 to 20 months after transplantation. Recent thymic emigrants and serum levels of IL-7 and IL-15 were similar within both groups of patients. Together, our results indicate that Homeostatic Peripheral Expansion (HPE) is the main mechanism to reach the normal numbers of CD8 T cells immediately after HSCT. A high and homogenous expression of MHC-I molecules is required for an optimal HPE.

Thrombotic thrombocytopenic purpura during immune reconstitution

The emergence of newer antiretroviral agents has revolutionized the treatment of patients struggling with multidrug-resistant strains of HIV. As a result, we are witnessing a higher incidence of immune reconstitution inflammatory syndrome, most commonly associated with mycobacterial, cryptococcal, herpetic and other infections, in addition to autoimmune conditions such as photodermatitis, systemic lupus erythematosus [1], Grave's thyroiditis [2] and sarcoid [3]. The pathogenesis of immune reconstitution inflammatory syndrome is unclear, but has been proposed to be a combination of thymic-independent homeostatic peripheral expansion, immune dysregulation between effector and regulatory T cells, increased inflammatory cytokines and dysregulated dendritic cells [4,5]. We present the case of a 48-year-old African-American man, with multiple drug-resistant HIV-1 infection, who was admitted to the hospital with acquired thrombotic thrombocytopenic purpura (TTP) 4 weeks after darunavir/norvir and enfuvirtide was added to tenofovir/emtricitabine. The patient had a past medical history only significant for chronic hepatitis B, with an undetectable viral load. After 4 weeks on the new regimen, his CD4 cell count increased from 94 (10%) to 351 (16.7%) cells/μl and his viral load decreased from more than 100 000 to 151 copies/ml. He presented with a non-productive cough, frontal headache, dark urine, dyspnea on exertion, myalgias and arthralgias from a previously asymptomatic state. Laboratory work-up (baseline in parenthesis) showed a hemoglobin level of 7.4 g/dl (11.2), platelets of 22 × 103/μl (221), lactate dehydrogenase 2450, creatinine 1.8 mg/dl (1.2), and a peripheral smear showed many schistocytes. An assay (Esoterix Inc., Austin, Texas, USA) showed the von Willebrand factor (vWF) cleaving protease to be deficient (< 8%), and the presence of a disintegrin-like and metalloprotease with thrombospondin-type 1 motif-13 (ADAMTS-13) vWF cleaving protease inhibitor (1.2 inhibitor units). No other inciting event was identified after extensive work-up. All his antiretroviral drugs were discontinued except for tenofovir/emtricitabine, whereas plasmapharesis, vincristine and prednisone were initiated. He had a full and uneventful recovery one month later. Subsequently, our patient was successfully restarted on antiretroviral therapy (darunavir/norvir and tenofovir/emtricitabine) without a recurrence of TTP, while experiencing increasing CD4 cell counts and decreasing viral loads. Although TTP has been described in relation to HIV [6], to our knowledge this is the first reported case of acquired TTP in the setting of immune reconstitution. Our patient had a low absolute CD4 cell count and CD4 cell percentage at baseline, followed by a ‘fulminant’ CD4 cell count recovery and viral suppression shortly after starting his new regimen. A Medline search failed to identify any association between TTP and antiretroviral agents, i.e. T-20, darunavir and others. Although a causal mechanism cannot be established from one case, we propose two insults accounting for a major alteration of vWF homeostasis based on previous descriptions of TTP [7]. The first alteration was caused by inflammatory cytokines (IL-6, IL-8, tumour necrosis factor alpha and others) that stimulate the ultralarge, hyperreactive vWF release from endothelial cells. The second insult is immune deregulation mediated by IL-6 and other autoantibodies that inhibit (ADAMTS-13) vWF cleaving protease activity, abrogating the ability of ADAMTS-13 to cleave ultralarge vWF, leading to the accumulation of ultralarge vWF in plasma and on the surface of endothelial cells. Could these two hits combined account for the high tendency for platelet aggregation and thrombosis that patients might be at risk of during the acute phase of immune reconstitution? The second reason we believe this case to be notable is its possible implication in the increase in cardiovascular events after immune reconstitution (unpublished). We believe that further studies should investigate the role of thrombosis in the increased cardiovascular events after acute immune reconstitution.

Interleukin-7 Produced by Antigen Presenting Cells Regulates the Homeostatic Peripheral Expansion of Naive CD4 T Cells.

Interleukin-7 (IL-7) is the only cytokine demonstrated thus far to directly support the development and maintenance of naive T cells. Interleukin-7 is produced mainly by the stroma of lymphoid organs but also to a lesser extent by antigen presenting cells. Dynamic changes in the relative availability of IL-7 during lymphopenia support T cell homeostatic peripheral expansion (HPE). Whereas HPE is very efficient at reconstituting the pool of mature CD8 T lymphocytes, regeneration of CD4 T lymphocytes via HPE is more problematic and deficits in CD4 T lymphocytes persist for years in humans following lymphodepletion. We demonstrate that in mice, CD4+ T cells undergo substantially diminished HPE compared to CD8+ T cells during lymphopenia. We hypothesized that CD4+ T cells uniquely require both MHC Class II plus IL-7 for survival and expansion during lymphopenia and that the availability of these signals are limited by the availability of IL-7 producing APCs. Consistent with this hypothesis, studies performed in chimeric mice showed that cells derived from APCs, but not stromal cells, provided the endogenous IL-7 that is require for naive CD4+ T cell HPE. Furthermore, whereas systemic exposure to high levels of rhIL-7 did not induce efficient homeostatic peripheral expansion of CD4+ cells, in vivo modulation of Class II expressing cells by flt3 ligand promoted homeostatic expansion of CD4+ T cells. Surprisingly however, we found that APCs respond to elevated systemic IL-7 by diminishing production of IL-7, a loop mediated by IL-7Rα and Stat5. As a result, elevated systemic IL-7 levels present during lymphopenia paradoxically diminish the production of IL-7 by APCs and thereby diminish CD4 HPE. This was directly demonstrated by experiments wherein CD4+ HPE was dramatically augmented in chimeric mice generated such that bone marrow derived cells lacked IL7Rα or Stat5. This work demonstrates a critical role for APC derived IL-7 in CD4+ regeneration and provides a new paradigm for understanding why prolonged CD4+ lymphopenia occurs following T cell depletion in humans.

Self Renewal and Proliferative Signals in the Bone Marrow Microenvironment Promote Homeostatic Peripheral Expansion of Donor-Derived T Cell Subsets Following Stem Cell Transplantation.

The bone marrow (BM), in addition to its role in providing hematopoietic stem cell support, is becoming increasingly recognized as a significant reservoir for T cells. Recent data have shown the BM to be the predominant site for homeostatic peripheral expansion (HPE). Constitutive HPE is a mechanism which allows maintenance of T cell numbers, while lymphopenia-induced HPE restores T cell numbers following T cell-depleting measures such as myelosuppression. While the role of BM in constitutive HPE in non-irradiated mice has been well documented, little is known of the role of BM in HPE following myeloablation. In the present study, we examined the contribution of BM to lymphopenia-induced HPE following myeloablative radiation conditioning and HSC transplantation, and documented the kinetics of expansion of donor-derived T cell subsets in the irradiated marrow and spleen. To this end, C57BL/6 and BoyJ congenic mice, differing in CD45 allelic expression, were used to track donor-derived T cell subsets using 8 color flow cytometry 5 days after transplantation of 10–20 × 10e6 low density BM cells (non-T cell depleted) into lethally-irradiated recipients (950cGy). At the time of analysis (d5 post transplant) the percentage of total donor cells in BM was approximately 20%, and 10% in the spleen. Of interest, an enhanced migration of CD8+ cells to BM was observed, increasing from 3±0.6% in steady state marrow to 8±3.3% of donor cells in transplanted mice. While only 10.1±1.0% of CD8+ cells in steady state BM exhibited a central memory phenotype (TCM, CD44hi, CD45RBhi, CD62Lhi), this percentage increased to 90±2.7% of donor CD8+ cells in day 5 transplanted BM (p<0.05). This preferential migration of TCM to irradiated BM is in contrast to the spleen, which contained approximately equal percentages of donor CD8+ naïve (CD44lo, CD62Lhi), effector (CD44hi, CD45RBlo, CD62Llo), and TCM cells. The reported increase in SDF-1 expression in irradiated BM relative to spleen may contribute to the enhanced TCM migration to BM, since TCM are more responsive to SDF-1 than naive cells. In experiments where donor cells were stained with CFSE prior to transplantation, proliferation of the different CD8+ subsets was followed. While the majority (∼97%) of BM TCM proliferated and lost CFSE fluorescence by day 5, a small percentage (2.8%) remained quiescent, exhibiting bright CFSE fluorescence (CFSEbright). In contrast, 10–20% of spleen TCM remained quiescent. These findings are in support of data documenting the BM as the predominant site for HPE, and illustrate the highly proliferative environment in the BM compared to spleen for expansion of TCM cells. Despite the higher degree of proliferation in the BM, 2-fold more BM CFSEbright TCM cells express IL7Ralpha, a molecule important in self renewal, compared to spleen CFSEbright cells. Given that TCM cells reportedly possess a similar self renewal genetic profile as hematopoietic stem cells, it is possible that TCM cells migrating through the BM following HSC transplantation are responsive to some of the same self renewal signals important for HSC self renewal, which would be presumably plentiful in the post-irradiation/transplantation BM microenvironment. In this regard, expression of IL7Ralpha by BM-homed TCM may allow for significant expansion while maintaining a self renewal phenotype, requirements which may be important for efficient HPE following myeloablation and stem cell transplantation.

GVHD Abrogates T Cell Responses to Dendritic Cell Vaccines but Not Vaccine-Induced Proliferation.

BACKGROUND: Allogeneic bone marrow transplant (BMT) is a potent form of immunotherapy against hematopoietic and potentially solid tumors, but relapse of malignancy and Graft versus Host Disease (GVHD) remain a major cause of morbidity and mortality. Dendritic cell (DC) vaccines represent a potential strategy to enhance the potency and efficacy of the Graft versus Tumor (GVT) effect without exacerbating GVHD, but little is known regarding the impact of the allogeneic BMT environment on vaccine responses. Previous work in our lab has shown that DC can effectively expand tumor-antigen specific T cells following minor-mismatched allogeneic transplantation, resulting in delay of tumor growth. However, GVHD completely abrogates these responses. We hypothesized that the mechanism behind the loss of DC vaccine responses was through diminished T cell proliferation in the setting of GVHD.METHODS: We established a minor histocompatibility antigen mismatched BMT model by transplanting B6 x C3H.SW (F1) thymectomized mice with either CD45.1+ B6 (allogeneic) or CD45.1+/45.2+ F1 (syngeneic) T cell-depleted bone marrow on day 0, followed by a donor lymphocyte infusion at day 14 to induce GVHD. On day 28, we adoptively transferred transgenic CD8+ (CD45.2, V beta 8.3+) or CD4+ (CD45.2, V beta 6+) T cells that only recognize components of the HY antigen to avoid cross-reactivity with allogeneic antigens. The T cells were labeled with CFSE prior to transfer to explore the impact of vaccine-induced proliferation in the presence or absence of GVHD.RESULTS: In the allogeneic BMT setting, both CD8+ and CD4+ HY-transgenic T cells given with a DC vaccine begin proliferating 5 days (CD8+ 40%, CD4+ 65% divided) after adoptive transfer. The syngeneic BMT vaccinated control mice show similar kinetics (CD8+ 38%, CD4+ 65% divided), suggesting GVHD does not decrease T cell proliferation. Interestingly, although HY transgenic T cells do not normally undergo homeostatic peripheral expansion (HPE) during lymphopenia, the CD4+ T cells proliferate in lymph nodes even in the absence of a vaccine (50% divided), but this was not observed in the respective syngeneic BMT unvaccinated control mice (0% divided).CONCLUSIONS: DC vaccines represent a strategy to improve the efficacy of the GVT response following allogeneic BMT, however, GVHD can abrogate DC vaccine responses. The negative impact of GVHD on vaccine responses does not appear to result from impaired DC vaccine-driven proliferation. Indeed, even in the absence of a vaccine, non-alloreactive CD4+ T cells proliferate via a mechanism that appears to be distinct from HPE. This data provides important insights towards optimizing antigen-specific vaccines administered in the allogeneic BMT setting.

Vaccination regimens incorporating CpG-containing oligodeoxynucleotides and IL-2 generate antigen-specific antitumor immunity from T-cell populations undergoing homeostatic peripheral expansion after BMT

Development of CD8(+) T-cell responses targeting tumor-associated antigens after autologous stem cell transplantations (ASCTs) might eradicate residual tumor cells and decrease relapse rates. Because thymic function dramatically decreases with aging, T-cell reconstitution in the first year after ASCT in middle-aged patients occurs primarily by homeostatic peripheral expansion (HPE) of mature T cells. To study antigen-specific T-cell responses during HPE, we performed syngeneic bone marrow transplantations (BMTs) on thymectomized mice and then vaccinated the mice with peptides plus CpG-containing oligodeoxynucleotides (CpGs) in incomplete Freund adjuvant and treated the mice with systemic interleukin-2 (IL-2). When CD8(+) T-cell responses were measured ex vivo, up to 9.1% of CD8(+) T cells were specific for tumor-associated epitopes. These large T-cell responses were generated by synergism between CpG and IL-2. When we injected mice subcutaneously with tumor cells 14 days after BMT and then treated them with peptide + CpG-containing vaccines plus systemic IL-2, survival was increased and tumor growth was inhibited in an epitope-specific manner. Depletion of CD8(+) T cells eliminated epitope-specific antitumor immunity. This is the first report to demonstrate that CD8(+) T-cell responses capable of executing antitumor immunity can be elicited by CpG-containing vaccines during HPE.

IFN-gamma Limits Peripheral T cell Expansion of Extrathymic T cells. (89.18)

Abstract When overexpressed under the p56Lck proximal promotor, Oncostatin M induces extrathymic T cell development in the lymph nodes. During LCMV infection, extrathymic (ET) T cells produce copious amounts of IFN-γ and their expansion is precocious but of limited amplitude because of early apoptosis. Moreover, ET T cells show significant apoptosis in uninfected mice. Hence, we sought to understand why ET T cells are more susceptible to apoptosis than thymic T cells. We found that the mitochondrial apoptotic pathway is likely involved in apoptosis of CD4+ ET T cells because of lower Il-7Rα and Bcl-2 levels compared to thymic T cells. In contrast, this deficit was not seen in CD8+ ET T cells. However, CD8+ ET T cells displayed higher Fas and active caspase 8 expression indicating apoptosis via the death receptor pathway. Moreover, we found that ET T cells and thymic T cells in homeostatic peripheral expansion (HPE) shared many similarities: memory phenotype, accelerated turnover and increased susceptibility to apoptosis. Moreover, spontaneous IFN-γ secretion by CD8+ ET T cells leading to massive accumulation in sera was observed. Finally, we found that CD8+ T cell survival was restored during HPE induced with ifngR−/−T cells as well as in ifng−/−ET T cells compared to IFN-γ-responsive T cells. Taken together, these results show that IFN-γ regulates apoptosis of ET T cells T cells in HPE and thereby limits their accumulation in vivo.