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The Journal of Heart and Lung Transplantation Volume 25, Number 2S

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DISCORDANT FUNCTION OF CD8 CD28 T CELLS IN THE BLOOD AND BAL OF LUNG TRANSPLANT RECIPIENTS: THE IMPACT OF CMV AND ACUTE REJECTION G.P. Westall,1 A.G. Brooks,2 T.C. Kotsimbos,1 1Heart and Lung Transplant Unit, Department of Allergy Immunology and Respiratory Medicine, Alfred Hospital, and Monash University Medical School, Melbourne, Victoria, Australia; 2Department of Microbiology and Immunology, Melbourne University, Melbourne, Victoria, Australia Following lung transplantation acute rejection and CMV pneumonitis have been associated with chronic rejection. Although alloreactivity and CMV reactivation represent opposing ends of the spectrum of immune dysregulation that occurs post-transplant, their potential effects on allograft function is largely dictated by the development of allo- and viral-specific T cell responses. Methods: In a prospective study of 19 lung transplant recipients (LTRs) we used flow cytometry to delineate CD8⫹ T cell subsets in both the blood and bronchoalveolar lavage (BAL). We related changes in T cell phenotype to both CD8⫹ T cell function (␥-IFN production) and to clinical status (episodes of acute rejection or CMV reactivation). Results: CMV seropositive LTRs had a high proportion of CD8⫹ T cells in the blood that were CD28 negative, suggestive of established T cell effector function. Additionally, in CMV naive LTRs there was a significant decrease in CD28 expression on blood CD8⫹ T cells following primary CMV infection. Functionally, the downregulation of CD28 on blood CD8⫹ T cells was associated with increased ␥-IFN production. A parallel analysis of BAL T cells revealed a CD8⫹CD28⫺ subset that became more prominent with time from transplant. In contrast to what was seen in the blood, the loss of CD28 on BAL CD8⫹ T cells was not associated with CMV reactivation, nor with increased ␥-IFN production, but conversely with a more favourable clinical outcome as reflected by fewer episodes of acute cellular rejection. Conclusion: We have demonstrated that changes in CD8⫹ T cell phenotype post-lung transplant relate to clearly defined changes in clinical status, and that these temporal changes are discordant when the two compartments of the blood and BAL are compared. The reduced ␥-IFN production by BAL CD8⫹CD28⫺ T cells and the association with reduced alloreactivity is suggestive of a tolerogenic subset that may identify a group of LTRs in whom immunosuppression can be safetly reduced. 322 P-GLYCOPROTEIN EXPRESSION ON PERIPHERAL BLOOD MONONUCLEAR CELLS AND BIOPSY PROVEN ACUTE REJECTION IN HEART TRANSPLANTATION J.B. Barnard,1 J. Fildes,1 S. Richardson,1 N. Khasati,1 V. Pravica,2 I.V. Hutchinson,2 C.T. Leonard,1 N. Yonan,1 1The Transplant Centre, South Manchester University Hospitals NHS Trust, Manchester, United Kingdom; 2Department of Immunology, Manchester University, Manchester, United Kingdom Objectives: P-glycoprotein (P-Gp), the membrane bound efflux pump encoded by the multi drug resistance (MDR1) gene may be one reason for poor response to immunosuppression in heart transplant recipients. This study set out to test the hypothesis that peripheral blood mononuclear cell expression of P-Gp is associated with increased levels of biopsy proven rejection, cyclosporine levels and nephrotoxicity in heart transplant recipients. Methods: Using a previously described flow cytometric method with directly conjugated monoclonal antibody to P-Gp, we assessed peripheral blood mononuclear cell expression of P-Gp in terms of mean fluorescence intensity in a group of 60 heart transplant recipients. 10 of the 60 patients were in the early post-operative phase. We tested

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for any correlation with biopsy proven rejection, difficulty obtaining Cyclosporine A (CsA) levels, and creatinine clearance. Results: There was a strong correlation between peripheral blood P-Gp expression and biopsy proven evidence of rejection p⬍0.001. This correlation remained when controlling for time and cyclosporine levels. There was no correlation between P-Gp and creatinine clearance, cyclosporine levels, or days post transplantation. Conclusions: High P-Gp expression on peripheral blood mononuclear cells is associated with an increased level of biopsy proven rejection independent of cyclosporine doses and serum trough levels and highlights the significance of the MDR1 gene in mediating heart transplant rejection. 323 THE ANTIMICROBIAL PEPTIDE, HUMAN BETA DEFENSIN-2, IN ACUTE REJECTION AFTER HUMAN LUNG TRANSPLANTATION R.L. Anderson,1 P.S. Hiemstra,1 I.A. Forrest,3 D. Proud,2 C. Ward,3 J. Lordan,3 P.A. Corris,3 A.J. Fisher,3 1Pulmonology Department, Leiden University Medical Centre, Leiden, Netherlands; 2 University of Calgary, Calgary, Canada; 3Applied Immunobiology and Transplantation Research Group, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom Human Beta Defensin-2 (hBD-2), an antimicrobial peptide, is released by airway epithelial cells on exposure to micro-organisms. In addition to antimicrobial activity, hBD-2 causes dendritic cell maturation and is chemotactic for T-lymphocytes. We hypothesised that hBD-2 may provide an important link between upregulated innate immunity and the adaptive T-cell responses seen in lung allograft rejection. Ross et al, showed recently in a small study that hBD-2 was elevated in bronchoalveolar lavage (BAL) of lung recipients with bronchiolitis obliterans syndrome compared to those with stable lung function (Transplantation 2004;78:1122–24). We measured hBD-2 by ELISA in BAL from a large cohort of lung recipients in a cross sectional study to determine the relationship between hBD-2, airway infection and acute rejection (AR). 70 lung recipients were investigated by BAL and transbronchial lung biopsy. 16 recipients were found to have organisms on culture of BAL fluid (12 Pseudomonas aeruginosa, 2 Aspergillus fumigatus, 1 Stenotrophomonas maltophilia, 1 Staphyloccocus aureus) and 54 were culture negative. Levels of hBD-2 were significantly increased in BAL of those culturing organisms, median (range) 1019 (0 –3490) pg/ml compared to those culture negative, 201 (0 –2500) pg/ml p⫽ 0.003 (kruskal-wallis). This was independent of the AR status. When only BALs from culture negative recipients were analysed, no difference in hBD-2 concentration was identified in 16 recipients with AR (grade A2 or higher) 0 (0 –321) pg/ml compared to 30 recipients with stable function, 55 (0 – 868) pg/ml, p⫽0.135. We conclude that hBD-2 is released by the lung allograft in response to micro-organisms but when those with pulmonary infection are excluded, AR is not associated with an increase in hBD-2 levels compared to recipients with stable function and no AR on biopsy. This suggests that allo-immune mediated graft injury after lung transplantation is not associated with increased hBD-2 production. 324 RESISTANCE TO DEPLETION AND ENHANCED HOMEOSTATIC PROLIFERATION BY MEMORY CELLS IN THE SETTING OF INDUCED LYMPHOPENIA D.C. Neujahr,1 C. Chen,1 L. Turka,1 1Medicine, University of Pennsylvania, Philadelphia, PA In human solid organ transplantation T cell depleting monoclonal and polyclonal antibodies have been used prolong allograft survival. Recent data in a murine cardiac allograft model demonstrated that the

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use of T cell depleting antibodies prior to a protocol of costimulatory blockade prevented tolerance induction. We hypothesized that the resistance to tolerance induction is related to the emergence of memory cells in the aftermath of lymphodepletion. The purpose of this study was to explore why memory cells predominate following depletion. We show that memory cells predominate shortly after subtotal lymphodepletion by two mechanisms: relative resistance to depletion and enhanced homeostatic proliferation. To demonstrated relative resistance to depletion, naive C57Bl/6 mice were treated with anti-CD4 and anti-CD8 antibodies to reduce total T cell numbers by 90%. We then observed using flow cytometry significant skewing of the remaining T cell pool toward a CD44hiCD45RBloCD62Llo phenotype before significant homeostatic proliferation had occurred. We then employed an adoptive transfer protocol in partially depleted mice given CFSE memory or naive cells to show that memory cells rapidly undergo multiple cell divisions, but the majority of transferred naive cells undergo fewer than two divisions in the same time. Regulatory cells (CD4⫹CD25⫹foxP3⫹) in contrast, were not significantly increased or decreased as a percentage of total remaining T cells, but were hypoproliferative relative to non-regulatory cells. Finally we show that the resistance to tolerance induction seen with subtotal T cell depletion can be overcome if additional regulatory cells are adoptively transferred at the time of transplant. We conclude that the resistance to tolerance induction seen following subtotal lymphocyte depletion can be attributed to alterations in the balance of naive, memory, and regulatory cells.

325 IMPACT OF CAMPATH-1H CONDITIONING ON DC SUBSETS IN PERIPHERAL BLOOD OF LUNG TRANSPLANT RECIPIENTS D. Metes,1 N. Murase,1 P. McGregor,1 A. Farran,1 C. Macedo,1 S. Yousem,1 J. Pilewski,1 D. Zaldonis,1 K. McCurry,1 A. Zeevi,1 1 Surgery, University of Pittsburgh, Thomas E. Starzl Transplantation Institute, Pittsburgh, PA Introduction: Dendritic cells (DC) play a key role in immunity and in tolerance induction. Recent publications have shown that immunosuppressive drugs may target and modulate DC function both in vitro and in vivo. In this study, we have prospectively analyzed in peripheral blood of lung transplant patients, the impact of Campath-1H preconditioning combined with FK-506 and low dose Prednisone maintenance therapy on DC subset frequencies and kinetics. Methods: Using multicolor flow cytometry, the percentage of DC precursor subsets Lin-DR⫹CD11c⫹ (myeloid) and Lin-DR⫹ CD123⫹ (lymphoid) were prospectively measured in peripheral blood of 46 lung transplant patients for minimum of 3 months up to 3 years. Peripheral blood DC subset values of 16 healthy individuals were used as control. Results: Overall, by 3 months post-transplantation the percentage of myeloid-derived Lin-DR⫹CD11c⫹ cells did not differ significantly in patients as compared with normal controls. However the percentage of Lin-DR⫹CD123⫹ cells was significantly lower (pⱕ0.008) in patients than in controls even at 3 years post transplant. Those patients who required additional corticosteroid treatment for acute rejection episodes (ⱖ2A) displayed a significant decrease in both myeloid (pⱕ0.03) and lymphoid (pⱕ0.02) DC counts compared with patients who remained rejection-free or patients with mild acute rejection (1A). Conclusion: These results indicate for the first time a significant preferential and prolonged depletion of Lin-DR⫹CD123⫹ DC subset in lung transplant patients receiving Campath-1H pre-conditioning. In addition, acute allograft rejection was associated with a decrease in both peripheral DC subsets.

The Journal of Heart and Lung Transplantation February 2006

326 DONOR BONE MARROW MODULATES THE HOST IMMUNE RESPONSES TO LUNG ALLOGRAFTS M. Pang,1 S. Li,2 S. Salgar,2 C. Liotta,2 R. Vazquez-Padron,2 J. Matthew,2 S.M. Pham,2 1Medicine, Univerisity of Miami School of Medicine, Miami, FL; 2Surgery, University of Miami School of Medicine, Miami, FL Aim: Recent studies show that donor bone marrow (BM) infusion reduces the incidence of obliterative bronchiolitis in lung recipients, and chronic rejection in renal recipients. However the mechanism(s) responsible for this observation is unknown. We sought to determine whether donor bone marrow infusion at the time of transplantation would have long-term effects on the host response to the lung allograft. Methods: Brown Norway (RT 1An) left lungs were transplanted into Lewis (RT 1A1) recipients. Some recipients (n ⱖ5 per time point) were given cyclosporine A(CsA, 10 mg/kg/day x 5 days, then 5 mg/kg/every other day until sacrifice), while others (n ⱖ5) received both CsA and 1.0 x 108 unmodified donor BM cells per rat on the day of transplant (CsA ⫹ BM). Animals were sacrificed at two weeks and 6 months posttransplant. Leukocytes from the bronchoalveolar lavage (BAL) fluid of the lung graft, native lung, peripheral blood, BM and thymus were collected. Four-color flow cytometry was used to analyse cell phenotypes. Results: At 6 months after transplant, only 4 of 11 lung grafts (36%) in the control versus 8 of 10 grafts (80%) in the BM-treated animals were accepted (p⬍0.05). At two weeks, BM infusion increased total donor-derived leukocytes (10.3% vs. 6.6%) and donor-derived dendritic cells (DC, 3.7% vs. 1.0%) in the graft. At 6 months, there was no difference in donorderived cell numbers or lineages. However, host derived CD4 (15.6% vs. 27.6%; p⫽0.08) and DC (4.5% vs. 9.1%; p⫽0.03) were reduced in lung grafts of BM-treated animals. Conclusion: Donor BM infusion increases the trafficking of donor cells to lung grafts in early stage, and decreases the infiltration of host CD4 and DCs in long-surviving grafts. These findings suggest that donor BM infusion has a long-lasting modulatory effect on host alloreactivity, resulting in improved graft survival. 327 EFFECT OF MHC CLASS II EXPRESSION ON CHIMERISM IN ALLOGENEIC FETAL LIVER TREATED MICE K. Tao,1 L. Mai,1 L.J. West,2 1Dept of Paediatrics, The Hospital for Sick Children, Toronto, ON, Canada; 2Dept of Pediatrics, Stollery Children’s Hospital, Edmonton, AB, Canada Introduction: Neonatal injection of allogeneic fetal liver cells (FLC) or semi-allogeneic bone marrow/spleen cells (F1BMSC) induces permanent acceptance of donor-type and third-party cardiac allografts. However persistent microchimerism could only be demonstrated after F1BMSC treatment, which also induces donor-type only skin graft survival, whereas FLC do not. This study examined the effect of recipient MHC expression on differential development of chimerism after neonatal tolerance induction. Methods: Neonatal C57BL/6 (B6; H-2b), MHC class I (CI⫺II⫹), class II (CI⫹CII⫺) and class I and II (CI⫺CII⫺) deficient mice (H-2b background) were injected with BALB/c (BALB; H-2d) FLC. At 8 wks, BALB cell persistence was sought by FACS analysis for H-2Dd expression. Additional treated mice were transplanted with BALB cardiac grafts. Results: BALB cells were not detected in FLC-treated B6 mice, nor in CI⫺II⫹ recipients. However, persistent chimerism was found in treated mice lacking MHC class II expression. In CI⫹CII⫺ mice, H-2Dd cells were 4.3– 8.5%; in CI⫺CII⫺ recipients, higher level of BALB cells was found: 14.1–27.4%. BALB heart grafts were accepted in FLC-