ALBERT

Description: Graft versus host disease (GVH) incidence and severity increases with older patient age. Host dendritic cells (DC) are responsible for initiating GVH, presumably as a result of activation at the time of transplant conditioning (as they are short lived after transplantation). Age-related alterations in host DC function have not been studied in a transplant setting. In this study, young (8–14 wk) and older (12–16 mo) mice were treated with the conditioning regimen of lethal irradiation (1100 cGy) and assessed at 6 h later for changes in DC costimulatory molecule expression and cytokine secretion. Older mice had slightly higher numbers of splenic classical DC (cDC; CD11chi Cl II+ B220−) and plasmacytoid DC (pDC; CD11clo Cl IIlo B220+) both before and after irradiation. No differences were observed in CD40, CD80, or CD86 expression between young and older mice, either before or after irradiation. IL-12, IFNγ, and TNFα producing cDC were more numerous in young untreated mice than in older mice. However, these cells decreased in young mice and increased in older mice after irradiation, resulting in generally higher numbers of cells producing these cytokines in older mice. IL-12, IFNγ, and TNFα producing pDC were more frequent in older mice than in young mice prior to irradiation, and both older and young mice showed increased frequency of cells producing these cytokines after irradiation. Overall, the highest numbers of cDC and pDC producing IL-12, IFNγ, and TNFα were present in older mice after irradiation. In both cDC and pDC populations, older mice had a higher frequency of IL-10+ cells prior to irradiation. After irradiation, young mice had a higher frequency of IL-10+ cells. These findings indicate that the balance of cytokine production in older mice after conditioning with irradiation is shifted to immunostimulatory cytokines rather than immunosuppressive cytokines. Murine GVH can be prevented by treatment with regulatory DC (rDC) in young animals (Sato et al., Immunity 2003). To determine whether this treatment is feasible in older animals, older BALB/c mice were transplanted with young C57Bl/6 BM. A subset of each group received age-matched host derived rDC. As expected, among mice that did not receive rDC, old recipients got more rapid and severe GVH than young recipients. This correlated with an increased frequency of residual host DC at the time of GVH in the older group. rDC were used to successfully treat GVH in older (as well as young) transplant recipients. These results provide the foundation for a cell based therapeutic approach to the treatment of GVH in older individuals.

Description: Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC, we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular, and percentages of CD34+ cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells, although present at normal frequencies within the CD34+ subset, were therefore absolutely decreased. In contrast, even the frequency of long-term culture-initiating cells within the CD34+ DC mPB cells was decreased, and the telomere lengths of these cells were also markedly reduced. Nevertheless, the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs.

Description: High dose therapy followed by autologous stem cell rescue for the treatment of multiple myeloma has been shown to be associated with improved outcome including increased complete response rates and survival. It is unclear what role, if any, autologous dendritic cell (DC) reconstitution patterns play in determining outcome. DC in peripheral blood (PB) can be divided into two subsets: DC1, which favor Th1 responses and DC2, which favor Th2 responses and in some cases appear to be immunosuppressive. In this companion study to a tandem stem cell transplant research protocol, PB DC subsets were studied just prior to the administration of mobilizing chemotherapy (baseline), at the time of stem cell collection, and on transplant d. 0, 15, 30, 60, and 90 following the first and second transplants. DC were identified as lineage (CD3/14/16/19) negative, HLA-DR+. Of these cells, DC1 = CD11c+CD123− or CD33hiCD4lo, and DC2 = CD11c−CD123+ or CD33lo/−CD4+. Precursor DC numbers (Pre-DC1 = CD45loCD34+HLA-DR+CD86+; Pre-DC2 = CD45loCD34+HLA-DR+CD10+) were also assessed in the stem cell products. GM-CSF (sargramostim) was used as part of the mobilization regimen as well as to enhance neutrophil recovery post-transplant. Twenty patients were enrolled and all are 〉90 d. post transplant #1. Six patients received a 2nd transplant. Median followup time is 25 months (range 3–56 months). Seven patients are in complete remission (CR), 7 have had a partial response, 4 have progressive disease, and 2 died of recurrent disease. In most cases, DC1 counts were higher than DC2 counts at all time points examined. At baseline, this was true for 70% of patients, indicating that immediately prior to transplant most patients maintain the DC1〉DC2 ratio found in healthy humans. DC subset numbers generally increased over baseline in mobilized PB, and correlated well with the patients’ baseline DC counts. Similarly, baseline DC subset numbers were highly correlated (R〉0.7) with the number of pre-DC1 or pre-DC2 found in the stem cell products. No patients with pre-DC110/ul) PB DC1 numbers at d. 90 consistently remain in CR. This study is the first to systematically examine DC subset reconstitution following autologous transplant for multiple myeloma. Our results provide preliminary evidence that the presence of high numbers of PB DC1 at d. 90 may predict a good long term prognosis, perhaps by promoting an effective endogenous immune response against residual disease. Furthermore, pre-DC1

Description: Abstract 3550 Poster Board III-487 Older patients experience increased severity of graft-versus-host disease (GVHD) following allogeneic bone marrow transplant and host dendritic cells (DC) are a vital initiator of this disease. To evaluate the effect of age on GVHD severity and establish a model that can be used to study treatment of GVHD, a complete MHC mismatched bone marrow transplant (BMT) model was used to induce GVHD. Older (≥14 month) and young (3-4 month) mice succumbed to GVHD, but older mice experienced faster onset of weight loss and earlier mortality. Prophylactic treatment with syngeneic (host strain-derived) regulatory DC (DCreg) has been shown to decrease mortality of GHVD in young mice (Immunity 2003 18: 367-379). To determine whether this treatment also decreases the morbidity from GVHD in young mice and to evaluate its efficacy in older mice, day +2 BMT mice were treated with DCreg (hereafter referred to as DCreg treated BMT mice). As previously described, DCreg were generated by culture of bone marrow cells (BM) with IL-10, GM-CSF, and TGF-β1 (Immunity 2003 18: 367-379). All DCreg treated mice exhibited a brief period of GVHD and survived at least 45 days post-BMT; overall, older mice had increased morbidity and decreased long-term survival than young mice. In vivo analysis of regulatory and co-stimulatory DC molecules (PD-L1, PD-L2, CD103, CD40, CD80, and CD86) on host and donor-derived DC revealed no statistical differences between young and older DCreg treated BMT mice. Paired Ig-like receptor B (PIR B) is an inhibitory receptor expressed on DC and GVHD is exacerbated in PIR B−/− mice (Nat. Immunol. 2004 5:623-629). An antibody that recognizes both PIR A and PIR B was used to look at expression on DC in DCreg treated BMT mice. PIR A/B expression was increased in vivo on young host DC compared to older host DC. Transferred DCreg are host-strain derived and it is unclear if the PIR A/B expression in these experiments was on residual host DC or the transferred DCreg. Future utilization of green fluorescent protein positive (GFP+) DCreg will determine the origin of upregulated PIR A/B expression in vivo. In contrast to cultured stimulatory DC, young cultured DCreg revealed increased PIR A/B expression and were negative for co-stimulatory DC molecule expression. This difference in PIR A/B expression potentially provides an explanation for the disparate outcome of older DCreg treated BMT mice. Previous studies have implicated Treg in the induction of tolerance in DCreg treated mice (Immunity 2003 18: 367-379). However, the mechanism of tolerance maintenance has not been investigated. Abrogation of Treg mediated suppression in vivo with anti-GITR antibody (DTA-1) did not result in recurrence of GVHD or mortality in young mice, implying maintenance of the GVHD-free state is not Treg mediated in young mice. Human transplant recipients have a compromised immune system from both the transplant and immunosuppressive drug regimen that results in increased susceptibility to infection. Upon infection, GVHD recurrence often occurs. A murine influenza infection model was used to assess the donor immune system's capacity to respond to infection and remain free of GVHD in young DCreg treated BMT mice. Forty-five days or more post-BMT, once completely recovered from GVHD and demonstrated to be allo-reconstituted, the DCreg treated BMT mice were intra-nasally infected with 0.1LD50 of the H1N1 mouse adapted A/PR/8/34 influenza virus. DCreg treated BMT mice did not have a resurgence of GVHD following infection and displayed similar morbidity to control mice. In conclusion, DCreg treatment of BMT mice results in decreased morbidity and mortality from acute GVHD in both young and older mice relative to untreated BMT mice. However, the older mice still do not fully recover and exhibit increased morbidity relative to young mice. The maintenance of tolerance observed in young DCreg treated BMT mice does not appear to be Treg mediated. DCreg treated BMT mice experience similar morbidity and mortality as control mice during influenza infection and do not experience a resurgence of GVHD, implying an intact immune response to influenza infection. Future studies will specifically examine the requirement of PIR B on DCreg for successful inhibition of GVHD. Additional work will continue to investigate the mechanisms of induction and maintenance of tolerance in DCreg treated BMT mice. S.M.S. is supported by the National Institutes of Health (grant #5T32 AI07485). Disclosures: No relevant conflicts of interest to declare.

Description: High dose therapy followed by autologous stem cell rescue for the treatment of multiple myeloma has been shown to be associated with improved outcome including increased complete response rates and survival. It is unclear what role, if any, autologous dendritic cell (DC) reconstitution patterns play in determining outcome. DC in peripheral blood (PB) can be divided into two subsets: DC1, which favor Th1 responses and DC2, which favor Th2 responses and in some cases appear to be immunosuppressive. In this companion study to a tandem stem cell transplant research protocol, PB DC subsets were studied prior to the administration of mobilizing chemotherapy (baseline), at the time of stem cell collection, on the day of transplant and at post transplant d. 15, 30, 60, and 90 following the first transplant. GM-CSF was used as part of the mobilization regimen as well as to enhance neutrophil recovery post-transplant. DC were identified as lineage (CD3/14/16/19) negative, HLA-DR+. Of these cells, DC1 were identified as CD11c+CD123− or CD33hiCD4lo, and DC2 were identified as CD11c−CD123+ or CD33lo/−CD4+. Sixteen patients have been enrolled and 11 are at least 90 d. post transplant #1. Median followup time is 12 months (range 7–25 months). 5 patients currently are in complete remission, 3 have had partial responses, 2 have stable disease, and 1 died of recurrent disease. In most cases, DC1 counts were higher than DC2 counts at all time points examined. At baseline, this was true for 77% of patients, indicating at baseline most patients maintain the higher DC1 numbers found in healthy humans. DC numbers increased in mobilized PB, and correlated well with the patients’ baseline counts. GM-CSF was more efficient at increasing DC2 than DC1, as has been reported for G-CSF. Post-transplant, DC recovery to at or near baseline levels occurred in most patients by d. 60. There was a trend for patients who are currently disease-free and those with partial responses to have a lower DC1:DC2 ratio (i.,e., DC2 reconstitution occurred more vigorously than DC1 reconstitution). The patient who died of progressive disease had the highest DC1 counts at several time points, and among the lowest DC2 counts leading to a consistently high DC1:DC2 ratio at 30, 60, and 90 days post-transplant. This finding suggests that in autologous transplant for multiple myeloma, proportionately increased numbers of DC2 are not associated with an increased risk of relapse, as was previously reported for allogeneic bone marrow transplant, but instead are associated with an improved likelihood of complete remission. Mobilization with a growth factor that favors the DC2 subset, such as GM-CSF, may promote the post-transplant production of a DC population that is able to assist in maintaining a complete response to high dose chemotherapy. If these trends are maintained upon final analysis of the patient population, additional means of selectively increasing DC2 numbers in this transplant setting would warrant further investigation.

Description: The mouse Lupo (I282N) mutation in proline-serine-threonine phosphatase–interacting protein 2 (PSTPIP2) leads to reduced expression of PSTPIP2 that is associated with a macrophage-mediated autoinflammatory disease. Another mutation in PSTPIP2, L98P, termed chronic multifocal osteomyelits (cmo), leads to a disease in mice that resembles chronic recurrent multifocal osteomyelits in humans. The cellular basis of cmo disease was investigated. cmo disease develops independently of lymphocytes and is cured by bone marrow transplantation. Macrophages, mast cells, and osteoclasts from cmo mice fail to express detectable PSTPIP2 protein. Asymptomatic Pstpip2cmo/cmo mice have increased circulating levels of macrophage inflammatory protein 1-α and interleukin-6, and their macrophages exhibit increased production of these inflammatory mediators, which is normalized by retroviral expression of wild-type PSTPIP2. Spleens of asymptomatic cmo mice contain increased numbers of macrophage precursors, and cmo mice mobilize more macrophage precursors in response to a sterile inflammatory stimulus. Signal transducer and activator of transcription 1 is elevated in cmo splenic macrophages, which also exhibit increased colony-stimulating factor-1–stimulated proliferation and increased extracellular signal-regulated kinase 1/2 phosphorylation. PSTPIP2 overexpression in macrophages leads to the opposite phenotype. Thus, PSTPIP2 deficiency causes both an expansion of macrophage progenitors and increased responsiveness of mature macrophages to activating stimuli, which together prime the organism for exaggerated and sustained responses leading to autoinflammatory disease.