correlation with cytomegalovirus infection and ...

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1991 78: 1373-1380

Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease P Reusser, SR Riddell, JD Meyers and PD Greenberg

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Cytotoxic T-Lymphocyte Response to Cytomegalovirus After Human Allogeneic Bone Marrow Transplantation: Pattern of Recovery and Correlation With Cytomegalovirus Infection and Disease By Pierre Reusser, Stanley R. Riddell, Joel D. Meyers, and Philip D. Greenberg The high rate of severe cytomegalovirus (CMV) disease after bone marrow transplantation (BMT) is related to the profound immunodeficiency posttransplant. Because cytotoxic T lymphocytes (CTL) have been implicated in resistance t o viral infections, we examined the restoration of the CMVspecific CTL response in 20 patients who received bone marrow from HLA-matched, CMV-seropositivedonors. Blood specimens were obtained from patients a t 1,2,and 3 months after BMT and from the donors on a single occasion. Peripheral blood mononuclear cells were cocultured with CMVinfected donor-derived fibroblasts for 2 weeks and then tested for cytotoxicity against CMV-infected and uninfected autologous or HLA-mismatched fibroblasts. Cytolytic activity was detected in all 20 donors, with specificity for autologous CMV-infected targets demonstrable in 17 (median CMV-specific lysis at an effector:target ratio of 15:l was 32%, range 18% t o 83%). Specific lysis was mediated by CDW,

class I-restricted T cells, as shown by inhibition with anticlass I monoclonal antibody and by selective depletion of effector cells. By contrast, CMV-specific CTL were detected in only 10 of 20 patients after BMT (median lysis 29% at 3 months post-BMT). None of these 10 patients developed CMV pneumonia, whereas 6 of the 10 patients with an undetectable CMV-specific CTL response after BMT died with CMV pneumonia. These results demonstratethat restoration of CMV-specific CTL responses may require an extended time period after BMT in some patients, and that such patients are at increased risk of developing severe CMV disease. Approaches t o reconstitute CMV immunity in BMT patients by adoptive transfer of CMV-specific CD8+ CTL clones derived from the bone marrow donor are now being pursued. 0 1991b y The American Society of Hematology.

I

may contribute to the high incidence of CMV infection and the development of severe CMV disease. Earlier investigations among BMT recipients have analyzed cytotoxic responses of lymphocytes obtained directly from the peripheral blood for CMV-infected target cells using long-duration cytotoxicity assay^.'^.'^ The lytic activity measured with this approach reflects several populations of cytolytic effector cells including both T cells and natural killer (NK) cell^.'^^^^^^* Thus, it was often not possible in these prior studies to distinguish the contributions to lytic activity of CD8+ class I major histocompatibility complex (MHC) restricted T cells from non-MHC restricted NK cell^.'^^'^ Furthermore, the use of more aggressive posttransplant immunosuppression in recent years could potentially alter the tempo of immunologic reconstitution to pathogens such as CMV." Efficient culture systems have been developed that permit in vitro activation and expansion of class I

NFECTION due to cytomegalovirus (CMV) represents a significant complication of bone marrow transplantation (BMT).',' After allogeneic BMT, CMV infection occurs in approximately 60% to 70% of patients who are CMV seropositive before transplant or who are seronegative but receive bone marrow from a seropositive donor, and is the principal infectious cause of death.' A fatal outcome from CMV infection is primarily related to the occurrence of interstitial pneumonia, which develops in one third of infected patients.'~~ Despite the development of new treatment regimens using antiviral drugs and Ig, CMV : % 0 5 pneumonia still has a fatality rate of approximately ' The natural history of CMV infection has been extensively studied. Following primary infection, immunocompetent hosts experience a mild or subclinical illness. The virus then enters a latent phase that usually persists for the life of the host, but CMV can reactivate and cause clinical disease if the host is immunosuppressed.' Animal models have been developed to elucidate the nature of the immune responses required to control active infection and maintain the virus in a latent state. Studies in murine models with murine CMV have suggested that CD8' CMV-specific cytotoxic T lymphocytes (CTL) are responsible for elimination of active infection and protective These results have been supported by inferential studies in humans."-'5 The peak incidence of CMV infection is during the first 3 months after BMT, and is thought to be related to the profound immunodeficient state induced by the treatment.I6'*l In light of the global immunologic dysfunction present during this time period, it has been difficult to precisely define the nature of the specific immunologic defects predisposing these hosts to CMV infection. However, BMT recipients demonstrate lymphopenia, with a diminished number of mature cytotoxic effector T cells, and exhibit impaired generation of new CTL effectors from lymphoid precursors in the early posttransplant period. As suggested by murine studies, this deficiency of CD8' T cells Blood, Vol78, No 5 (September 1). 1991: pp 1373-1380

From the Fred Hutchinson Cancer Research CenteG and the Division of Infectious Diseases and Departments of Medicine and Immunology, University of Washington, Seattle. Submitted December 12,1990; acceptedApril 30,1991. P.R. was supported by grants from the Swiss National Research Foundation and Swiss Cancer League. S.R.R. is a Fellow of the Leukemia Society ofAmerica and a recipient of an American Cancer Society of Clinical Oncology Young Investigator Award. The work was supported in part by Grants CAI8029 andAI27757from the National Institutes of Health. Presented in part at the 2nd Intemational Cytomegalovirus Workshop, San Diego, CA, March 27, 1989, and at the Annual Meeting of the American Society for Cancer Research, Washington,DC, May 23, 1990. Address reprint requests to Pierre Reusser, MD, Department of Intemal Medicine, University Hospital, CH-4031, Basel, Switzerland. The publication costs of this am'cle were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C.section I734 solely to indicate this fact. 0 1991 by TheAmerican Society of Hematology. 0006-4971f91f7805-0001$3.0ofo 1373

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REUSSER ET AL

monitored ~ e e k l y . 2Due ~ to the seropositive donor status, the MHC antigen-specific T cells, so that it is possible to patients were transfused with blood products that were not evaluate the presence of all potentially virus-reactive T screened for antibody to CMV. Sixteen of the 20 recipients were cells, not just circulating lymphocytes exhibiting direct lytic CMV seropositive before transplant, with 14 of 16 receiving CMV activity. These methods have been used to detect and prophylaxis to day 30 after transplant with high-dose intravenous characterize CMV-specific CD8’ CTL in CMV-seroposiacyclovir,Mand 2 of 16 receiving prophylactic foscamet until day 75. tive subjects with no evidence of active CMV infe~tion,’‘~~~~” Expansion of CMV-spec@c CTL in vitro. CMV-specific CTL and should be applicable to an analysis of the presence of reactivity in patients was evaluated at 1, 2, and 3 months after CMV-specific CTL in BMT recipients. transplant and compared with the response in marrow donors. A In this prospective study of BMT donor-recipient pairs, modified culture system,” based on previously described methwe have used sequential in vitro stimulation of peripheral od~,”,~‘ was developed to expand CMV-specific CI‘L in vitro. blood lymphocytes with autologous CMV-infected fibroBriefly, skin biopsies were obtained from each marrow donor to establish fibroblast lines for use as both stimulator and target cells. blasts to analyze seropositive marrow donors for the presFibroblast lines were grown in Waymouth’s medium supplemented ence of class I MHC-restricted CMV-specific CTL and with 20% fetal calf serum (FCS), and used between passages 4 and their BMT recipients for the development of a CMV12. The fibroblasts were tested before use for mycoplasma contamspecific CTL response after transplant. The results of these ination by DNA hybridization (Mycoplasma T.C. 11; Gen-Probe, studies were correlated with the occurrence of CMV Inc, San Diego, CA). Autologous fibroblasts were plated in 6-well infection and CMV disease in the BMT recipients during plates at 0.5 x l@cellsiwell, and infected for 2 hours with the the first 3 months after transplant. The data suggest that human CMV strain AD 169 (American Type Culture Collection, generation of class I-restricted CTL responses to CMV may Rockville, MD) at a multiplicity of infection (MOI) of 5:l before greatly influence the capacity of the host to limit the initiation of lymphocyte culture. CMV strain AD 169 was initially severity of CMV infection after BMT. propagated in human foreskin fibroblasts, and the infectivity of the MATERIALS AND METHODS

Patient population. The investigation was conducted prospectively among 20 allogeneic marrow transplant recipients and their healthy marrow donors. The patients were selected for study if they had a human histocompatibility leukocyte antigen (HLA)identical, mixed lymphocyte culture-nonreactive sibling marrow donor seropositive for CMV IgG antibody. Informed consent was obtained from both the patients and the marrow donors, and the study protocol was approved by the institutional review board of the Fred Hutchinson Cancer Research Center (Seattle, WA). Characteristics of the study population are summarized in Table 1. The methods of BMT and posttransplant care have been reported previously?8 Each patient received cyclosporine plus a short course of methotrexate as prophylaxis for graft-versus-host disease (GVHD), and trough serum cyclosporine levels were Table 1. Characteristicsof the 20 Marrow Donors and Recipients

Median age, in years (range) Sex (male/female) Underlying disease: Acute nonlymphocytic leukemia Acute lymphocytic leukemia Chronic myelogenous leukemia Hodgkin’s lymphoma Non-Hodgkin‘s lymphoma RAEB in transformation Pretransplantconditioning regimen: Cyclophosphamide/FTBI VP-I6/BCNU/cyclophosphamide Busulfan/cyclophosphamide PretransplantCMV serology: Positive Negative Patients alive at 3 mo after transplant

Donors

Recipients

39 (18-54) 11/9

40 (17-56) 11/9 9 2 5 1 2 1 15

3 2 20 0

16 4* 14

Abbreviations: RAEB, refractow anemia with excess of blasts; FTBI, fractionated total body irradiation. *Two of these four patients seroconverted after transplant.

resultant virus stocks used for the study was 3 x lo6plaque-forming units/mL. Peripheral blood mononuclear cells (PBMC) were obtained from whole blood containing preservative-free sodium heparin (Lymphomed, Inc, Rosemont, IL) by Histopaque (Sigma, St Louis, MO) gradient centrifugation. The cells were resuspended in RPMI-HEPES supplemented with 10% CMV seronegative human AB serum and 5 x lo-’ mol, 2-mercaptoethanol (Sigma), and dispensed at 10’ cellsiwell in the 6-well plates containing autologous CMV-infected fibroblast stimulators. After 1 week of incubation at 37°C in a humidified 5% CO, atmosphere, the cultured cells were harvested, washed, and recultured at a ratio of 20:l with fresh CMV-infected fibroblast stimulators in 6-well plates with autologous irradiated (3.5 Gy) PBMC as filler cells. Fortyeight hours after restimulation, half of the culture supernatant was replaced by fresh medium containing recombinant interleukin-2 (rIL-2; Hoffmann-LaRoche, Inc, Nutley, NJ) to achieve a final concentration of 2 U/mL. This culture system resulted in preferential persistence and expansion of CD8’ T cells with CMV-specific cytolytic acti~ity?~.” Cytotoxici@ assay. The panel of target cells used for each cytotoxicity assay included autologous and HLA-mismatched CMVinfected and uninfected fibroblasts. All fibroblast lines used as HLA-mismatched targets were lysable by autologous effector cells. Fibroblast targets were preincubated before use with recombinant interferon-y (rIFNy; Schering, Inc, Kenilworth, NJ) for 48 hours at 800 U/106 cells, because this has been shown to increase the sensitivity and specificity of the assay for CMV-specific CTL by enhancing HLA-class I antigen expression.z Fibroblast targets were labeled with ”Cr (100 pCi/106 cells; New England Nuclear, Boston, MA), and an aliquot was infected overnight with CMV AD 169 at an MOI of 5:l. Fibroblasts were then tIypsinized, suspended at 10s cells/mL in 10% FCS, and 100 p L (lo4 cells) dispensed in triplicate into 96-well round-bottom plates together with 100 pL of effector cell suspensions at an effector:target (E:T) ratio of 15:l. Higher E T ratios were also evaluated when enough lymphocytes were available. Although more lysis was detected at higher E:T ratios, it did not improve the ability to detect CMV-specific CTL in donors or patients. To confirm that specific lysis was class I-restricted, cytotoxicitywas also assayed against targets preincubated for 20 minutes at 22°C with 25 &mL of the anti-HLA class I monoclonal antibody (MoAb) W6132 (a gift of Dr D. Geraghty, Fred Hutchinson Cancer Research Center).” After incubation of

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CTL RESPONSE TO CMV AFTER MARROW TRANSPLANT

the plates for 4 hours at 37°C in a humidified 5% CO, atmosphere, 100 JLLof supernatant was harvested from each well, and radioactivity was measured in a y counter. Specific lysis was calculated by the standard formula, with maximum release reflecting counts per minute (cpm) from incubation of targets with 1% Nonidet P40solution (Sigma), and spontaneous release, which never exceeded 30% of maximum release, reflecting cpm from targets incubated with medium alone. There was no difference in spontaneous or maximum release between infected and uninfected targets. Selective depletion of effector cells. To determine the phenotype of CMV-specific CTL, effector cells were suspended in RPMI at a concentration of 6 x lo6cells/mL, incubated on ice for 45 minutes with 2.5 &mL of either MoAb OKT4 or OKT8 (Ortho Diagnostics, Inc, Raritan, NJ), washed, and incubated with rabbit complement (lot no. 3588; Pel Freeze, Inc, Brown Deer, WI) at a dilution of 1:3 at 37°C for 30 minutes. Phenotypic analysis by flow cytometry before and after MoAb and complement treatment demonstrated selective and greater than 95% effective depletion of CD4' and CD8' T-cell subsets, respectively. Lymphoproliferativeassay. The lymphoproliferative response to CMV antigen and to phytohemagglutinin (PHA) was determined at the time CTL cultures were initiated. PBMC were suspended at lo6 cellsimL in RPMI with 20% FCS and 100 JLLdispensed into wells of 96 round-bottom plates. CMV antigen was prepared by glycine-extraction'' and tested at three dilutions (1:100, 1:400, and 1:1,600).Because optimal responses were detected at a dilution of 1:400, only these results are presented. PHA (Difco Laboratories, Detroit, MI) was used at a concentration of 10 &mL. One hundred microliters of CMV antigen or PHA was added to triplicate wells containing PBMC, and the plates incubated at 37°C in a humidified 5% CO, atmosphere for 96 hours. Sixteen hours before harvest, 1 pCi of 'H-thymidine (New England Nuclear) was added to each well. Wells were collected using a semi-automated harvester, and samples measured in a p-scintillation counter. Results are expressed as a stimulation index calculated by dividing the mean cpm of cells exposed to CMV antigen or to PHA by the mean cpm of cells incubated with medium alone. A stimulation index of 4 or greater was considered to indicate a positive lymphoproliferative resp0nse.9~ Virologic surveillance. Virologic surveillance before transplant and during the first 100 days after transplant consisted of viral cultures from throat, urine, and blood performed at least weekly. Specimens were inoculated onto monolayers of foreskin fibroblasts and observed for 4 weeks for cytopathic effects. Serologic surveillance consisted of weekly testing of serum samples for IgG antibody to CMV by enzyme-linked immunosorbent assay (CMV Stat; Whittaker M.A. Bioproducts, Walkersville, FL). Dejinition of CMV infection and disease. CMV infection was defined as the presence of CMV in clinical specimens by culture or by histology. CMV disease was defined as evidence of CMV in tissue specimens, and in the case of CMV pneumonia in bronchoalveolar lavage, associated with clinical symptoms and signs. Statistical analyses. Continuous variables were compared by using the Wilcoxon rank-sum test and dichotomous variables by Fisher's exact test. Pvalues < .05 were considered significant. RESULTS

CMV-specificCTL among marrow donors. Lytic activity for CMV-infected targets was demonstrable in all 20 CMV-seropositive marrow donors, with CMV-specificCTL activity readily detectable in 17 (85%) (Fig 1). Median specific lysis of autologous CMV-infected targets was 32% and was significantly higher than the low level of background lysis detectable with autologous uninfected or

100-

80-

I* p, .... 2

..

I

.. ......

-15

n

I

I

Inf

Uninf

Autologous

i I'

ii:: I

:

i m.. I

Uninf Inf HLA- mismatched

Fig 1. Cytolytic activity of cells from 17 CMV-seropositive bone marrow donors with demonstrable CMV-specific CTL. Cytotoxicity was assayed at an E:T ratio of 15:l against autologous CMV-infected (Inf) and uninfected fibroblast targets (Uninf), as well as against HLA-mismatched CMV-infected and uninfected fibroblast targets (insufficient effector cell numbers precluded testing against HLAmismatched uninfected fibroblast targets in two marrow donors). Lysis of the autologous CMV-infected targets was significantly higher than lysis of each of the other three targets (P < .OW1 by Wilcoxon rank-sum test). Median indicated by horizontal bar.

HLA-mismatched CMV-infected or uninfected targets (P < .0001). The preferential lysis of autologous CMVinfected targets over HLA-mismatched infected targets suggested that this culture system was generating classical CD8' class I MHC-restricted CTL.This was further analyzed by preincubating the targets with anti-class I MoAb, which reduced the lysis of autologous infected targets by an average of 46% (P = .002) (Table 2). Moreover, selective depletion of CD8' T cells abrogated CTL activity, whereas depletion of CD4' T cells had no effect (Table 2). Specific lytic activity was not demonstrable in 3 of 20 seropositive marrow donors. Although the median lysis of autologous CMV-infected targets with effector cells from these three donors was 45% (range 21% to 55%), these effector cells exhibited similar levels of lytic activity against autologous uninfected or HLA-mismatched targets. Thus, this nonspecific lytic activity obscured the detection of any CMV-specificCTL activity potentially present. CMV-specificCTL activity after transplant. Ten marrow recipients (50%) developed a detectable class I MHCrestricted CMV-specific CTL response within the first 3

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1376 Table 2. Effects of (IClass IMoAb Blocking or Selective Depletion of T-cell Subsets on Cytotoxicity of CMV-Specific T-cell Cultures From Three CMV Seropositive Subjects Percent Specific Lysis of Targets at E:T of 15: 1'

Subject

1 2 3

Untreated Effector and Target

Target Preincubated Effector Treated With With N Class I MoAb C Alone aCD4 C uCD8

+

28

14

47

25 21

36

33 39

5

26 38 38

41

+C

4 0

Abbreviations: a,anti; C, complement. 'Lysis of autologous CMV-infected fibroblasts targets. Lysis of autologous uninfected and HLA-mismatched CMV-infected and uninfected targets was