Nancy G. Higgins

Phenotypic characterization of normal human placental mononuclear cells

The placenta is a rich source of immunocompetent cells. We have studied the phenotype, number and origin of placental mononuclear blood cells isolated from 32 normal term placentae using 4 color flow cytometry. Respective maternal and cord blood leucocyte preparations were also compared. Placental tissue without extraembryonic membranes was cut into small pieces and divided. One portion was washed extensively with ice-cold PBS. Both tissue portions were disrupted in a blender and cells were dissociated by using a 180 mu sieve. Leucocytes were isolated by Ficoll-Hypaque density gradient centrifugation. Maternal and cord bloods were HLA typed and in cases of HLA-A2 or B7/40 disparity, monoclonal anti-HLA antibodies to these antigens showed that unwashed placental tissue contained 35% maternal and 65% fetal cells. This ratio, however, was not reflected for a given cell phenotype. In comparison, washed placental tissue contained cells of fetal origin only. Both unwashed and washed placental tissue contained fewer CD3 and CD4, but more CD8 cells than maternal and cord blood. Markers of NK cells such as, CD16, CD56, and CD57 showed this cellular phenotype to be 15 times more abundant in the placental preparations than in cord and maternal blood. The quantitative differences between peripheral blood and placental CD8 and NK cells were further explored with an antiprogesterone receptor antibody in combination with anti-CD8, anti-CD57 and anti-HLA-DR. The number of progesterone receptor (PGR) positive cells was three times higher in placental tissues than in cord or maternal blood. These data indicate that the phenotypic frequencies of certain placental leucocytes are significantly different from maternal and fetal peripheral blood. Progesterone and the presence of PGR may be important in the differential retention of placental leucocytes.

Utilization of intravenous immunoglobulin to ameliorate alloantibodies in a highly sensitized patient with a cardiac assist device awaiting heart transplantation. Fluorescence-activated cell sorter analysis.

Surgery surrounding the use of mechanical assistance in cardiac transplant candidates often leads to multiple blood/platelet transfusions and subsequent development of alloantibodies. This is a case report of a 50-year-old male patient who had received blood transfusions during coronary bypass grafting 9 years earlier. He presented in acute and chronic heart failure and, despite therapy, became moribund with multisystem organ failure. His ejection fraction was 10%. A Novacor ventricular assist device was implanted on May 19, 1995 (day 0). The patient received 44 U of blood and 20 U of platelets. Although his percent reactive antibodies (PRA) were negative before surgery by fluorescence-activated cell sorter analysis, the PRA 3 days after implantation of the ventricular assist device was 80%; it increased to 100% by day 7. In an attempt to decrease the PRA, intravenous immunoglobulin was given at 3-week intervals. The PRA became negative and the patient received a donor heart that was negative by fluorescence-activated cell sorter cross-match on day 64. On days 69-72, a dramatic increase in alloantibody activity was promptly reversed with additional intravenous immunoglobulin. Currently at posttransplant month 12, the patient shows no humoral, cellular, or vascular evidence of rejection.

The strength of donor-specific antibody is a more reliable predictor of antibody-mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients.

Mujtaba MA, Goggins W, Lobashevsky A, Sharfuddin AA, Yaqub MS, Mishler DP, Brahmi Z, Higgins N, Milgrom MM, Diez A, Taber T. The strength of donor‐specific antibody is a more reliable predictor of antibody‐mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients.
Clin Transplant 2011: 25: E96–E102.

Analysis of anti-HLA antibodies in sensitized kidney transplant candidates subjected to desensitization with intravenous immunoglobulin and rituximab.

Background Preexisting donor-specific antibodies against human leukocyte antigens are major risk factors for acute antibody-mediated and chronic rejection of kidney transplant grafts. Immunomodulation (desensitization) protocols may reduce antibody concentration and improve the success of transplant. We investigated the effect of desensitization with intravenous immunoglobulin and rituximab on the antibody profile in highly sensitized kidney transplant candidates. Methods In 31 transplant candidates (calculated panel-reactive antibody [cPRA], 34%–99%), desensitization included intravenous immunoglobulin on days 0 and 30 and a single dose of rituximab on day 15. Anti–human leukocyte antigen antibodies were analyzed before and after desensitization. Results Reduction of cPRA from 25% to 50% was noted for anti–class I (5 patients, within 20–60 days) and anti–class II (3 patients, within 10–20 days) antibodies. After initial reduction of cPRA, the cPRA increased within 120 days. In 24 patients, decrease in mean fluorescence intensity of antibodies by more than 50% was noted at follow-up, but there was no reduction of cPRA. Rebound occurred in 65% patients for anti–class I antibodies at 350 days and anti–class II antibodies at 101 to 200 days. Probability of rebound effect was higher in patients with mean fluorescence intensity of more than 10,700 before desensitization, anti–class II antibodies, and history of previous transplant. Conclusions The desensitization protocol had limited efficacy in highly sensitized kidney transplant candidate because of the short period with antibody reduction and high frequency of rebound effect.

Crossmatch-positive liver transplantation in patients receiving thymoglobulin-rituximab induction.

Background Positive crossmatch (CM) in liver transplantation (LT) is associated with worse outcomes. Role of induction immunosuppression in this setting remains to be studied. Methods One thousand consecutive LT patients receiving rabbit antithymocyte globulin±rituximab induction were studied. Pretransplantation sera of 55 CM-positive (CM+) patients were tested for C1q-fixing donor-specific antibodies (DSA). Diagnosis of antibody-mediated rejection required presence of diffuse vascular C4d expression on liver biopsies. Results CM was positive in 112 (11%) recipients. Antibody-mediated rejection was observed in 3 (0.03%) patients, whereas acute cellular rejection (ACR) occurred in 31 (3%) patients. CM+ status was associated with a higher incidence of ACR (9% in CM+ vs. 2% in CM-negative [CM−]; P<0.01) and chronic rejection (4% in CM+ vs. 1% in CM−; P<0.01). Graft survival was slightly lower in CM+ patients (at 1 year; 85% in CM+ vs. 89% in CM−; P=0.26). Patients with autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis as a group had a tendency toward CM+ status as well as developing ACR. Upon multivariate analysis, CM status was the strongest predictor of ACR (B=1.14; P=0.02). Only half of CM+ patients harbored C1q-fixing DSA. Presence of C1q-fixing DSA was not associated with increased incidence of ACR. Conclusions In LT, CM+ status is associated with an increased incidence of acute rejection, chronic rejection, and slightly worse graft survival. With the use of rabbit antithymocyte globulin±rituximab induction, overall low rejection rates can be achieved in CM+ LT.

Detection of IGA anti-OKT3 antibodies in OKT3-treated transplant recipients.

Murine OKT3 monoclonal antibodies function as an immunosuppressant drug for organ transplant recipients. A contraindication to retreatment may develop, however, because a high proportion of OKT3-treated patients form anti-OKT3 antibodies. Previous data have shown that only antiidiotypic IgG antibodies can negate the beneficial effect of the drug. Eighty-two OKT3-treated transplanted patients were tested by ELISA for IgG, IgK and IgA anti-OKT3 antibodies and compared with 200 controls. The anti-OKT3 antibody-positive sera were screened additionally by flow cytometry for the presence of antiidiotypic activity by measuring Ortho OKT3-FITC activity on a CD3-positive cell line, Jurkat, before and after incubation with serial dilutions of patient and control sera. Forty-four of 82 patients developed antibodies to OKT3, 20 manifested IgG, 20 produced both IgG and IgA, and 4 IgA only. We never detected IgM anti-OKT3. Of the 44 anti-OKT3-positive patient sera, 25 showed antiidiotypic specificity. Two IgG/IgA anti-OKT3-positive patient sera were IgG-depleted by Protein G. Both continued to exhibit antiidiotypic IgA activity. IgA anti-OKT3 was associated with low serum OKT3 levels and lack of ability of OKT3 to lower total CD3 cell numbers to therapeutic levels. This is the first report of IgA anti-OKT3 antibody in transplant recipients. Isotype IgA anti-OKT3 was observed in 54% of the patients whose sera tested positive for anti-OM by ELISA. The IgG/ IgA anti-OKT3-positive patient sera tested continued to exhibit antiidiotypic OKT3 reactivity when depleted of IgG. We urge that OKT3-treated patients be monitored routinely for IgA anti-OKT3 antibodies to avoid the expense and potential complications of retreatment with this drug in sensitized patients.