Control of cytomegalovirus in bone marrow transplantation chimeras lacking the prevailing antigen-presenting molecule in recipient tissues rests primarily on recipient-derived CD8 T cells

Abstract

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule Ld were transplanted into the Ld gene deletion mutant BALB/c-H-2(dm2), an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of Ld-positive donor-derived and Ld-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against Ld. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by Ld within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.

FIG. 1

Establishment of chimerism in an HvG setting of MHC class I-mismatched BMT after incomplete lymphohematoablative conditioning. BALB/c-H-2^dm2 recipients (uninfected [BMT] and infected with murine CMV on the day of BMT [BMT + CMV]) were γ irradiated with a sublethal dose of 6 Gy and then reconstituted with the indicated graded numbers of BMC derived from BALB/c donors. (A) Kaplan-Meier survival plots for groups of 20 mice. The asterisk marks the conditions of BMT under which all subsequent experiments were performed. (B) Origin of BMC in the repopulated recipient BM measured for 6-week survivors. Femoral BMC of three mice were pooled for analysis. Positive expression of L^d identifies donor (BALB/c)-derived BMC. The histograms are based on the analysis of 10,000 BMC. RCN, relative cell number. Expression of L^d (abscissa) is given in log FITC fluorescence (FL-1) intensity. (C) Analysis of chimerism among T cells and T-cell subsets derived from the spleens of 6-week survivors. Lymphocytes derived from three spleens were pooled for analysis. Individual testing of aliquots taken before forming the pools gave essentially the same results (not depicted). Three-color fluorescence (FL) analyses were performed for the marker combinations FITC (FL-1)-L^d plus PE (FL-2)-TCR α/β plus RED613 (FL-3)-CD4 or RED613 (FL-3)-CD8. A first gate was set on lymphocytes; a second gate was set on α/β T cells positive in FL-2 (top), on CD4 T cells simultaneously positive in FL-2 and FL-3 (center), and on CD8 T cells simultaneously positive in FL-2 and FL-3 (bottom). FL-1 histograms are shown for 10,000 gated cells. For chimeric cell populations, the percentage of donor-derived, L^d-positive cells is indicated. Controls of D^d expression in the chimeras and of L^d expression in BALB/c-BALB/c isochimeras gave single peaks with positive FL-1 (not shown); these controls were made to make sure that we were not misled by MHC class I-negative cells and by L^d-low/high-expressing cells, respectively.

FIG. 2

Preferential recruitment of CD8 T cells to the infected lungs and control of pulmonary infection. (A) Three-color cytofluorometric analysis of lung infiltrate lymphocytes in the combination FITC (FL-1)-CD8 plus PE (FL-2)-TCR α/β plus RED613 (FL-3)-CD4. A lymphocyte gate was set in the FSC-versus-SSC plot, representing cell size and cell granularity, respectively (upper left); a second gate was set on positive FL-2 to select cells expressing TCR α/β (lower left). (Right) Expression of CD4 and CD8 among the FL-2-positive α/β T cells, shown in a two-dimensional dot plot of FL-3 and FL-1 fluorescence, respectively. The percentages of CD4 and CD8 T cells as well as the CD8/CD4 ratio are indicated. The analysis of the subpopulations was based on 20,000 α/β T cells. (B) Kinetics of lung infiltration. For independent but analogous transplantations, CD8/CD4 ratios within pulmonary α/β T cells were determined in weekly intervals. Each closed circle represents the analysis at the indicated time point of a particular transplantation. Data refer to a pool of pulmonary infiltrate cells derived from five recipients. The median values for the independent transplantations are indicated by dashes; the shaded area indicates the range of CD8/CD4 ratios observed in uninfected recipients under otherwise corresponding conditions. n.t., not tested. (C) Kinetics of murine CMV replication in the lungs. Virus titers in the lungs of infected recipients were determined for the transplantations shown in panel B. Closed circles represent the median values of the virus titers of three recipients per transplantation and time point. The median values for the independent transplantations are marked by dashes. The dotted line indicates the detection limit (DL) of the plaque assay. n.t., not tested.

FIG. 3

Two-color immunohistological analysis of liver tissue infection and T-cell infiltration. Pulmonary lymphocytes derived from lung infiltrates of infected chimeras at 5 weeks after BMT were tested for their antiviral and protective ability by intravenous adoptive transfer into infected BALB/c indicator recipients that were immunocompromised by 6 Gy of γ irradiation. The analysis was performed on day 14 after cell transfer. Group A, controls given no pulmonary T cells; groups B, C, and D, recipients of adoptive transfer of 10⁴, 10⁵, and 10⁶ pulmonary lymphocytes, respectively. Panels A1 through D3 represent liver sections from individual recipients, three per experimental group. Infected cells, mostly hepatocytes, are identified by red nuclear staining of the IE1 protein pp89 of murine CMV. Infiltrating T cells are identified by black staining of CD3ɛ. Arrows mark areas of interest shown in overview (A3 through D3) and resolved to greater detail (A3* through D3*). Note the cytomegalic cells with prominent intranuclear inclusion bodies in panel A3* and the inflammatory foci visible under the conditions of group C. Photographs cover 0.08 mm² (overview panels) and 0.005 mm² (detail panels) of liver sections. The bar markers represent 50 μm.

FIG. 4

Inverse relation between T-cell infiltration and infection of liver tissue. The two-color immunohistology shown in Fig. 3, groups A to D, was analyzed quantitatively by counting infected, IE1 protein-expressing hepatocytes and infiltrating CD3ɛ-expressing T lymphocytes for representative 10-mm² areas of liver sections. Symbols represent median values, and bars indicate ranges of the counted cell numbers for three recipients per experimental group.

FIG. 5

The antiviral function of pulmonary infiltrate-derived lymphocytes is accomplished by CD8-positive effector cells. The dose-dependent antiviral effect of pulmonary lymphocytes upon adoptive transfer of graded cell numbers is documented for the spleens (top) and lungs (bottom) of infected BALB/c indicator recipients immunocompromised by 6 Gy of γ irradiation. The antiviral T-cell subset was identified by adoptive transfer of 10⁶ pulmonary lymphocytes depleted of either CD8 or CD4 T cells by treatment with anti-CD8 (αCD8) or anti-CD4 (αCD4) MAb and complement, respectively. Closed circles represent virus titers per organ for three recipients per experimental group. The median values are marked by dashes; each dotted line indicates the detection limit (DL) of the plaque assay.

FIG. 6

Chimerism among CD8 T cells in pulmonary infiltrates. Pulmonary lymphocytes were isolated at the peak of infiltration after BMT (6 Gy, 10⁷ BMC) and infection. Three-color cytofluorometric analysis was performed for the combination FITC (FL-1)-D^d or FITC (FL-1)-L^d plus PE (FL-2)-TCR α/β plus RED613 (FL-3)-CD8. A gate was set on lymphocytes, and the analysis was restricted to α/β T cells by a second gate set on positive FL-2. (A and C) Two-dimensional dot plots of FL-3 (CD8) versus FL-1 (D^d [A] and L^d [C]) for 20,000 α/β T cells. CD8-positive cells are marked by a frame. (B and D) Histograms of FL-1 for the framed FL-2- and FL-3-positive CD8 T cells. The percentages of L^d-negative, recipient-derived and L^d-positive, donor-derived cells are indicated.

FIG. 7

Absence of MHC class I L^d-specific, HvG-directed cytolytic activity in the lungs of mixed chimeras. Pulmonary infiltrate lymphocytes isolated at the peak of lung infiltration were tested for ex vivo cytolytic activity at the indicated effector-to-target (E/T) cell ratios on target cells expressing L^d (A; parental P815 mastocytoma), L^d plus B7-1 (B; transfectant P815-B7), and L^d plus Fas (C; transfectant P815-Fas). Insets show the cytofluorometric analyses verifying the expression of B7-1 and Fas by the respective transfectants, as well as their absence on parental P815. FL, log FITC fluorescence intensity. The total CTL activity contained in the tested lymphocyte population was determined by the TCR redirected lysis assay using a MAb directed against TCR α/β bound to Fc receptors that are expressed constitutively by P815 (D). All target cells expressed surface L^d as detected by cytofluorometry, and all were lysed by a CTL line specific for L^d (not shown).

FIG. 8

Cytolytic activity of sorted recipient-derived and donor-derived CD8 T cells. (A) Pulmonary lymphocytes were isolated at the peak of infiltration, and CD8-positive cells were enriched by immunomagnetic selection. (Top) The efficacy of the positive selection was monitored for an aliquot of the cells by a two-color cytofluorometric analysis of CD8 (PE, FL-2) versus L^d (FITC, FL-1) expression performed for 20,000 cells with no preselection of gates. (Bottom) The majority of the CD8 T cells were labeled for L^d only and were then sorted into L^d-negative and L^d-positive sets. The efficacy of the sorting was monitored for 2,000 cells of each set by two-color cytofluorometric analysis of TCR α/β (PE, FL-2) and L^d (FITC, FL-1) expression performed with no preselection of gates. (B) The cytolytic activity of pulmonary CD8 T cells was determined by TCR α/β redirected lysis at the indicated effector-to-target (E/T) cell ratios. (Left) Cytolytic activity of the chimeric CD8 T-cell population after immunomagnetic purification but before the cytofluorometric sorting; (right) cytolytic activity of the sorted cells. For comparison, the presort activity is included for the part of the titration marked by dotted lines.

FIG. 9

Control of viral replication in tissues of adoptive transfer recipients by sorted donor-derived and recipient-derived pulmonary CD8 T cells. The in vivo antiviral activity of the sorted CD8 T cells was tested by adoptive transfer of 50,000 cells into immunocompromised, γ-irradiated (6 Gy) indicator recipients, which were chosen to be isogenic either with the original BMT donor (BALB/c) or with the original BMT recipient (BALB/c-H-2^dm2). Virus replication in liver and spleen of the indicator recipients was monitored on day 14 after the cell transfer by ISH specific for a gB gene DNA sequence of murine CMV. The red staining visualizes infected cells identified by the accumulation of viral DNA in an intranuclear inclusion body. Each photograph covers a 0.05-mm² area of tissue section. The bar marker represents 50 μm.

FIG. 10

MHC genotype-specific, differential antiviral function of sorted donor-derived and recipient-derived pulmonary CD8 T cells. The results of the histological ISH analyses shown in Fig. 9 for selected 0.05-mm² areas of tissue were substantiated by counting the numbers of infected cells in representative 10-mm² areas of liver and spleen sections. Closed circles represent median values, and bars indicate ranges of the counted cell numbers for three adoptive transfer recipients per experimental group.