Absolute Number Of Lymphocytes Research Articles

Lymphocyte subpopulations in the cerebrospinal fluid and peripheral blood in multiple sclerosis.

Subpopulations of lymphocytes in the CSF and peripheral blood were studied in 30 patients with MS, 16 with other neurological diseases (OND) and 15 control subjects without any neurological abnormalities. In patients with relapse of MS, the absolute numbers of total lymphocytes, alpha-naphthyl acid esterase (ANAE) positive, E-rosette forming and bearing the "avid" FcIgG receptor lymphocytes were significantly increased in the CSF as compared with stable or slowly progressive MS patients, patients with other OND and control subjects. The relative number of ANAE-positive cells was higher, and "avid"FcIgG receptor bearing cells lower in the CSF of all patients with MS than in the two other groups. The significance of the finding is unclear. The imbalance between lymphocyte subpopulations may reflect a primary defect in MS, or may be secondary, due to the presence of circulating immune complexes. In peripheral blood no substantial differences in lymphocyte behavior were observed between MS patients and other groups.

Kinetics of Regulatory T Cells in the Ovalbumin Asthma Model in the Rat

Background: The kinetics of regulatory T cells (T_Reg) in allergic diseases such as asthma are only partly known. Methods: The asthma model in the Fischer rat with ovalbumin (OVA) sensitization and aerosol challenge was used. The relative and absolute numbers of leukocytes, lymphocytes and T_Reg subsets were determined by flow cytometry in the lung interstitium and draining bronchial lymph nodes at different time points after two challenges, and lung function was tested in parallel. Results: The progressive number of challenges resulted in increased relative and absolute numbers of lymphocytes and in particular of T_Reg. The T_Reg number was augmented with each aerosol challenge and was already significantly increased 6 h after the second challenge. The relative (%) and absolute numbers of CD4+ and CD8+ T_Reg and dendritic cells showed different kinetics after two challenges. The leukocyte numbers in the lung did not correlate with lung function. Conclusion: T_Reg increased surprisingly early after challenge in the lung tissue. Relative and absolute numbers of leukocyte subsets should always be calculated. The kinetics of different leukocyte subsets can only be determined when several time points are studied.

Multivariate Analysis of Prognostic Factors in CLL: A Study on 431 Patients Showing Usefulness of Novel Biological and Old Clinical Parameters in Predicting Shorter Survival: An Italian Multicentric Study

B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease with highly variable clinical courses. Recently, besides classical clinical parameters, including Rai/Binet's staging system, novel biological factors acquired prognostic relevance especially for risk-adapted therapeutic strategies: IgVH mutational status, ZAP-70 and CD38 expression, chromosome aberration (del13q, trisomy 12, del11q, del17p) detected by FISH analysis. In a recent report, Wierda et al constructed a prognostic nomogram predictive for overall survival based on clinical and routine laboratory characteristic of 1674 CLL patients: age, beta2 microglobulin, absolute lymphocyte count, sex, Rai stage and number of involved lymph node groups. Biological prognostic features were not considered in this model but the authors concluded that “future works will focus on incorporation of new prognostic factors into this prognostic model. Actually, unlike other haematological malignancies, there is no a standard Prognostic Index that can be used to group CLL patients according clinical and new biological prognostic parameters and to guide the choice of treatment. To assess which factors give independent contribution in predicting shorter survival we collected 784 patients data from 4 laboratories. All variables were measured at diagnosis or before start of therapy; they included: age above 65 years, male sex, Rai stage >0, beta2 microglobulin >2.5 mg/L, absolute lymphocyte count (ALC), involvement of 3 or more nodal groups, high risk FISH cytogenetics (17p- or 11q-), IgVH mutational status, ZAP-70 and CD38 expression. Survival was measured from diagnosis until death from any cause. A sample of 431 patients was obtained after deleting cases with missing values. Median follow-up was 60 months (54–65). There were 63 deaths (15%) during follow-up, with a not reached median survival at 250 months. During follow-up 43% of patients were treated. Cox proporzional hazard model was used for univariate and multivariate analysis. P <0.05 was considered to be statistically significant. Multivariate model was developed including those variables statistically significant in univariate analysis and further variables were eliminated from the final model by backward selection. IgVH, ZAP-70, CD38, ALC, male sex were not significant in univariate analysis. In multivariate analysis the following variables predicted for shorter survival (table 1): age>65 years, Rai stage>0, beta2>2.5mg/L, high risk FISH. In a larger subset of patients (568), selected with all variables except beta2 microglobulin, the above results were confirmed, in particular no independent contribution was given by IgVH, ZAP-70 and CD38, even if they turned out significant in univariate analysis. Beta2 microglobulin and high risk FISH or only high risk FISH retained prognostic power respectively in a subset of 198 patients under 65 years of age and in a subset of 252 patients with Rai stage 0. In conclusion age>65 years, B2 microglobulin> 2,5 mg/L, Rai stage >0, and high risk FISH abnormalities (del 17p or 11q) will be considered by clinician because of independent prognostic factor of OS. A nomogram will developed to predict for survival using the independent covariates identified in multivariate analysis. This weighted prognostic model will permit to obtain a total point score predictive for median survival of each patient, based on clinical and biological characteristic.Table 1: MULTIVARIATE ANALYSIS (COX MODEL, p = Wald test)Variableshazard ratio (confidence interval)p-valueAge>652.19 (1.26–3.79)0,0052Nodal sites involved >= 3rrBeta-2 microglobulin >2.5mg/L1.87 (1.06–3.28)0,03Rai stage > 02.13 (1.26–3.62)0,0051FISH 17p- or 11q-2.41 (1.42–4.08)0,0011r = removed by bacward selection

Impaired Immune Reconstitution after HLA-Mismatched Transplantation Due to Antigen Specific T-Cell Recognition Restricted by the Disparate Haplotype

The applicability of HLA matched hematopoietic stem cell transplantation (SCT) is limited by the lack of HLA matched donors. In contrast, an HLA haploidentical donor can be found for almost all patients. Posttransplantation immunodeficiency leading to high morbidity and mortality from viral infections is a major barrier to the broader application of haploidentical SCT. In HLA mismatched SCT profound T cell depletion is necessary to prevent GVHD, but also leads to slower T-cell reconstitution. Vigorous T-cell depletion, however, does not fully explain the increased immunodeficiency. Despite a gradual normalization of absolute T-cell and total lymphocyte number after haploidentical SCT, the immunodeficiency may persist for years. Additionally, an increase in the number of mismatched HLA alleles between patient and donor leads to more profound post transplantation immunodeficiency. We hypothesized that another explanation for the long lasting immunodeficiency is T-cell antigen recognition via the haplotype disparate between patient and donor. T-cells recognizing viral epitopes in the donor non-shared HLA molecule will attack virus infected hematopoietic cells of donor origin but not virus infected non-hematopoietic cells of patient origin. To investigate the role of T-cell disparate haplotype restriction in posttransplantation immunodeficiency, we characterized the immune responses against CMV in two patients at various time points after haploidentical and mismatched SCT. The first patient was HLA-A2 negative and transplanted with SCT of an HLA-A2 positive haploidentical donor. After the SCT he suffered from life threatening CMV reactivations for almost two years. Using tetramers composed of the HLA-A2 molecule and CMV-pp65-NLV epitope, we determined that in this patient CMV specific T-cells restricted to the donor HLA-A2 were present from 6 months after the SCT and remained present during the two year period of recurrent CMV infections. Since the HLA-A2 allele was not shared between patient and donor, these results indicate that T cells restricted by non-shared donor-HLA were unable to protect against infections. The second patient transplanted with an HLA mismatched SCT, suffered from CMV reactivations only in the first 6 months after SCT. Already 3 months after SCT CMV specific T cells restricted to the shared HLA-B8 allele were present in high concentrations, whereas the CMV specific T-cells restricted to the non-shared donor HLA-A1 allele disappeared in time. Our results suggest that for an effective immune response the T cells need to recognize viral epitopes restricted to the HLA shared between patient and donor and that impaired immune reconstitution after haploidentical SCT is, in part, the result of antigen specific T cell responses restricted to the disparate haplotype.

Laboratory control values for CD4 and CD8 T lymphocytes. Implications for HIV-1 diagnosis.

With the advent of standard flow cytometric methods using two-colour fluorescence on samples of whole blood, it is possible to establish the ranges of CD3, CD4 and CD8 T lymphocyte subsets in the routine laboratory, and also to assist the definition of HIV-1-related deviations from these normal values. In 676 HIV-1-seronegative individuals the lymphocyte subset percentages and absolute counts were determined. The samples taken mostly in the morning. The groups included heterosexual controls, people with various clotting disorders but without lymphocyte abnormalities as well as seronegative homosexual men as the appropriate controls for the HIV-1-infected groups. The stability of CD4% and CD8% values was demonstrated throughout life, and in children CD4 values less than 25% could be regarded as abnormal. The absolute counts of all T cell subsets decreased from birth until the age of 10 years. In adolescents and adults the absolute numbers (mean +/- s.d.) of lymphocytes, CD3, CD4 and CD8 cells were 1.90 +/- 0.55, 1.45 +/- 0.46, 0.83 +/- 0.29 and 0.56 +/- 0.23 x 10(9)/l, respectively. In patients with haemophilia A and B the mean values did not differ significantly. In homosexual men higher CD8 levels were seen compared with heterosexual men and 27% had an inverted CD4/CD8 ratio but mostly without CD4 lymphopenia (CD4 less than 0.4 x 10(9)/l). However, some healthy uninfected people were 'physiologically' lymphopenic without having inverted CD4/CD8 ratios. When the variations 'within persons' were studied longitudinally over a 5-year period, the absolute CD4 counts tended to be fixed at different levels. As a marked contrast, over 60% of asymptomatic HIV-1+ patients exhibited low CD4 counts less than 0.4 x 10(9)/l together with inverted CD4/CD8 ratios. Such combined changes among the heterosexual and HIV-1-seronegative homosexual groups were as rare as 1.4% and 3%, respectively. For this reason, when the lymphocyte tests show less than 0.4 x 10(9)/l CD4 count and a CD4/CD8 ratio of less than unity, the individuals need to be investigated further for chronicity of this disorder, the signs of viral infections such as HIV-1 and other causes of immunodeficiency.

Evaluation of the Immune System in Rats and Mice Administered Linear Ammonium Perfluorooctanoate

Repeated high doses of ammonium perfluorooctanoate (APFO) have been reported to affect immune system function in mice. To examine dose-response characteristics in both rats and mice, male CD rats and CD-1 mice were dosed by oral gavage with 0.3-30 mg/kg/day of linear APFO for 29 days. Anti-sheep red blood cell (SRBC) IgM levels, clinical signs, body weights, selected hematology, and lipid parameters, liver weights, spleen, and thymus weights and cell number, selected histopathology, and serum corticosterone concentrations were evaluated. In rats, linear APFO had no effect on production of anti-SRBC antibodies. Ten and 30 mg/kg/day resulted in systemic toxicity as evidenced by decreases in body weight gain to 74 and 37%, and increases in serum corticosterone levels to 135 and 196% of control, respectively. In mice dosed with 10 and 30 mg/kg/day, marked systemic toxicity and stress were observed, as evidenced by a loss in body weight of 3.8 and 6.6 g, respectively (despite a tripling of liver weight), approximately 230% increase in serum corticosterone, and increases in absolute numbers of peripheral blood neutrophils and monocytes with an accompanying decrease in absolute lymphocyte numbers. Immune-related findings at 10 and 30 mg/kg/day that likely represent secondary responses to the systemic toxicity and stress observed at these doses include: decreased IgM antibody production at 10 (20% suppression) and 30 mg/kg/day (28% suppression); decreased spleen and thymus weights and cell numbers; microscopic depletion/atrophy of lymphoid tissue at 10 (thymus) and 30 mg/kg/day (spleen). In summary, no immune-related changes occurred in rats, even at doses causing systemic toxicity. In mice, immune-related changes occurred only at doses causing significant and profound systemic toxicity and stress.

In vivo study of toxoplasmic parasitemia using interferon-γ-deficient mice: Absolute cell number of leukocytes, parasite load and cell susceptibility

Toxoplasma gondii infection was induced in wild type (WT) C57BL/6 and BALB/c mice and same-background interferon-γ knockout (GKO) mice by peroral inoculation of Fukaya strain cysts. We studied parasitemia, absolute cell number of leukocytes, and cell susceptibility in peripheral blood leukocyte (PBL) subsets in vivo. Parasitemia was observed in both WT and GKO mice, although the pattern of the parasite load was totally different among them. After infection, the absolute number of neutrophils in peripheral blood increased in both GKO C57BL/6 and GKO BALB/c mice with statistical significance, while it rose up slightly in susceptible WT C57BL/6 mice, but declined moderately in resistant WT BALB/c mice. The absolute number of lymphocytes in both WT and GKO mice decreased significantly after infection. Although leukocyte number per μl decreased significantly in both strains of WT mice, it increased in GKO BALB/c mice. There were no differences in susceptibility of PBL subsets to T. gondii infection as assessed by quantitative competitive polymerase chain reaction in both WT and GKO mice. These results indicate that each of the PBL subsets was infected in vivo. The increase of neutrophils only among immunocompromised or susceptible strains suggests that the neutrophil may be involved in the lethal process of T. gondii infection. The lack of any difference in cell susceptibility per μg gDNA among the PBL subsets examined implies that the neutrophil may contribute to the whole body dissemination process of the parasite in vivo more than other PBL subsets through the increase in number.

Activity of Nuclear Factor of Activated T Cells Is Independent of the Number of Peripheral Lymphocytes in FTY720-Treated Patients

We studied the inhibition of nuclear factor of activated T-cell (NFAT)-regulated gene expression (interleukin-2 [IL-2], interferon-γ [IFN-γ], granulocyte-macrophage colony-stimulating factor [GMCSF]) in cyclosporine (CsA)-treated de novo patients with and without lymphopenia due to FTY720. Materials and Methods Sixteen CsA-treated de novo renal transplant recipients received either FTY720 (n = 8) or mycophenolic acid (MPA; n = 8) in combination with low-dose steroids. Expressions of IL-2, INF-γ, and GMCSF were measured at 1 (visit 1), 2 (visit 2), and 14 (visit 3) months postoperatively using peripheral lymphocytes obtained at 0 hour versus 2 hours after CsA intake. Gene expression was assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Results CsA 0- and 2-hour levels were comparable in both groups. Absolute NFAT-regulated gene expression was significantly lower among FTY720-treated patients at visits 1 and 2. Median residual NFAT-regulated gene expression was 5.9% in the FTY720 group and 4.2% in the MPA-treated group at visit 1, increasing to 7.2% and 7.0%, respectively, at visit 3. One borderline rejection occurred in the MPA group. Median serum creatinine was 1.5 mg/dL among FTY720 and 1.8 mg/dL among MPA patients. Conclusions Despite significantly lower expression of NFAT-regulated genes, the relative reduction in NFAT gene expression was comparable in both groups. The absolute number of lymphocytes was not relevant for this immune response. In addition, gene expression increased to comparable levels after FTY720 was switched to MPA. The relative residual gene expression increased with reduction in CsA dose.

Relationship of Lymphocyte-Neutrophil Ratio to Heterophile Antibody Result

Abstract The heterophile antibody test (HAT) is commonly ordered at the same time as a complete blood cell (CBC) count in cases of suspected infectious mononucleosis. An absolute lymphocytosis with the presence of greater than 10% atypical lymphocytes is a good predictor of a positive HAT. We retrospectively studied patients who underwent HAT and had a simultaneous CBC count during an 8.5-year period. The ratio of the absolute number of lymphocytes to the absolute number of neutrophils (L/N) was determined to see if this measure could be useful in distinguishing those most likely of having a negative HAT. A total of 5104 patient samples were analyzed of which 501 (9.8%) were HAT positive. The L/N ratio was less than 0.5 in 2214 patients (43.4%), and in this group, only 46 (2.1%) were HAT positive. In the group whose L/N ratio was greater than or equal to 0.5, the HAT positivity rate rose to 15.7% (455/2888), for a relative increase of 60%. In the HAT-positive group, 153 (30%) did not demonstrate an absolute lymphocytosis. Restricting HAT to those having a CBC count with an L/N ratio greater than or equal to 0.5 will increase the HAT positivity rate and lead to a reduction of HAT performed.

Varicella Zoster Virus–Specific Immune Response after Treatment with Sodium Stibogluconate for Cutaneous Leishmaniasis

Sodium stibogluconate has been associated with the reactivation of varicella zoster virus (VZV) in otherwise healthy adults who receive the drug as treatment for cutaneous leishmaniasis. Ten patients receiving daily sodium stibogluconate underwent phlebotomy at baseline and at day 10. Flow cytometry-based immunophenotyping, VZV-specific IgG levels, and lymphocyte proliferative responses and intracellular cytokine secretion to VZV, cytomegalovirus, tetanus toxoid, superantigen, and mitogens were performed at both time points. The absolute number of total leukocytes, total lymphocytes, and lymphocyte subsets decreased overall without predilection for any particular subset of lymphocytes, such that the percentage of the total lymphocyte population for each lymphocyte subset did not change significantly (except for a marginal increase in percentage of cytotoxic T cells). Antibodies to VZV were measured in seven patients before and after treatment, and did not change. Lymphocyte proliferative responses to VZV and other antigens and mitogens did not change from baseline. The mechanism for the increased rate of VZV reactivation after treatment with sodium stibogluconate remains undefined.

A dopamine mechanism is implied in the acquisition and expression of amphetamine and stress-induced effects observed in the lymphocyte subpopulations

Drugs of abuse and stress are associated with changes in circulating cell populations and reductions in cell-mediated immune responses. The main goal of this study was to determine the influence of repeated and acute d-amphetamine treatments on the foot-shock stress-induced effects on the peripheral lymphocyte subpopulations, and the involvement of a dopamine mechanism in the development and expression of this phenomenon. Wistar rats received an acute (5 mg/kg/day i.p.) or a repeated (2 mg/kg/day i.p. during 9 days) amphetamine treatment, and were exposed to a foot-shock stress (1 mA, 3 s) 4 days after the last amphetamine injection. Another group was administered with haloperidol (1 mg/kg/day i.p.) 15 min previous to each daily amphetamine injection or previous to the foot-shock stress session. Then, blood cells stained with monoclonal antibodies against CD3-FITC, CD8-PE and CD4-Cy-Chrome, and against CD161a-FITC, CD3-PE, and CD45RA-Cy-Crhome, were analyzed by multiparameter flow cytometry. The exposure to a foot-shock stress induced a decrease in the absolute number of peripheral lymphocytes, as well as in CD4+ and CD8+ T-cells and B-cells in acute and repeatedly amphetamine-treated rats, whereas the NK-cell population remained unchanged. Haloperidol administration previous to each drug administration or the foot-shock stress session reversed these effects. This study provides strong evidence that dopamine can play a more general role in the influence of amphetamine on the stress-induced effects on the lymphocyte subsets.

Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

IntroductionWhile consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation.MethodsIn a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content.ResultsErythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not vary with changes in erythrocyte n-3 LCPUFA levels, but the iodination reaction was reduced with higher n-3 LCPUFA content.ConclusionThe data show that regular long-term consumption of n-3 enriched foods leads to lower numbers of NK cells and neutrophil iodination activity but higher lymphotoxin production by lymphocytes. These changes are consistent with decreased inflammatory reaction and tissue damage seen in patients with inflammatory disorders receiving n-3 LCPUFA supplementation.

Lymphocyte subsets in peripheral blood of patients with moderate-to-severe versus mild plaque psoriasis

In several studies peripheral blood T-cells have been quantified, yet few data are available on lymphocyte subsets in moderate-to-severe psoriasis (in terms of extent and activity of lesions) versus mild psoriasis. The objective is to compare lymphocyte subsets in peripheral blood of patients with moderate-to-severe disease (PASI-score ≥12) to patients with mild disease (PASI-score <12) and to healthy subjects. By means of flow cytometry method, lymphocytes in peripheral blood of 27 patients with psoriasis and 10 healthy controls were characterized. The absolute number of total lymphocytes was markedly decreased in patients with moderate-to-severe psoriasis as compared to patients with mild disease and normal subjects. Cellcounts of all analysed subsets were found to be increased in more severe psoriasis, except for CD8+CD45RO+ cells. The under-representation of CD8+CD45RO+ cells is compatible with the dynamics of acquired immunity, which requires a time log after the relapse of the lesions to differentiate from CD45RA+ naive cells.

Significant alterations in peripheral blood lymphocyte subsets in patients with somatoform disorder.

Previous studies have suggested that somatoform disorders (SFD) might be associated with changes in the function of the central and autonomic nervous systems. The aim of this study was to examine the possible immunological differences between SFD and healthy controls. Twenty-four patients with SFD and 13 healthy individuals completed the psychological questionnaires to assess symptom reporting [Symptom Checklist-90 Revised (SCL-90-R)] and to diagnose for SFD [Screening for Somatoform Symptoms scale (SOMS-scale)]. Participants also provided a blood sample taken in the morning, which was analysed with an automated cell counter to determine the number of leucocytes per μl and with flow cytometry to determine lymphocyte subsets. With the exception of a higher T4/T8 ratio in the patient group, which was mainly because of lower CD8 counts, there were no significant differences in the absolute number of lymphocytes (subsets) between patients with SFD and healthy subjects. A positive correlation between B-lymphocyte subsets (CD19+CD22+, CD19+CD5+, CD19+CD3-) to all scales of the SCL-90-R, except somatisation, were found in SFD. Additionally, a positive correlation was found in SFD between CD14+CD16+ monocytes and somatisation (0.573) on the SCL-90-R scale. These data indicate that patients with SFD have an enhanced humoral immunity as shown by increased B-cell numbers and furthermore an elevated T4/T8 ratio because of lower CD8 suppressor cells. Further studies will be required to determine whether these alterations in lymphocyte subsets are directly involved in the pathophysiology of SFD.

SGN-33, Lintuzumab, Demonstrates Anti-Leukemic Activity in Preclinical Models of AML.

SGN-33 is a humanized IgG1 antibody with signaling properties that also mediates effector functions including Antibody-Dependent Cellular Cytotoxicity (ADCC). SGN-33 targets CD33, a sialoadhesion family member expressed on acute myeloid leukemia (AML) cells, eliciting complete remissions in some AML patients. In the laboratory, SGN-33 mediates natural killer (NK) cell lysis of both multi-drug resistant (MDR- positive) and MDR-negative AML cell lines. Additionally, SGN-33 significantly increases survival in SCID mouse disseminated models of MDR-negative and MDR- positive AML. Immunomodulatory compounds such as thalidomide and its analog, lenalidomide (Revlimid®), have been shown to enhance NK cell function and anti-cancer activity. In particular, Revlimid in combination with rituximab demonstrated greater tumor cell killing compared to the antibody alone. Given these reports, we investigated whether thalidomide or lenalidomide would enhance the anti-tumor effects of SGN-33. In vitro, lenalidomide augmented NK function in a dose-dependent fashion, manifested as significant increases in perforin expression. Thalidomide and lenalidomide enhanced ADCC activity (% maximal specific lysis) while having no significant effects on tumor cell levels of CD33. In vivo, treatment of SCID mice with lenalidomide resulted in a 2-fold increase in the absolute numbers of lymphocytes (including NK cells). In vivo studies evaluating the efficacy of the combination of SGN-33 and lenalidomide in SCID mouse models of AML are in progress. In summary, our studies demonstrate the anti-leukemic activity of SGN-33 against MDR-positive AML cell lines, suggesting that this unconjugated anti-CD33 antibody may yield benefit in clinical settings where other therapies fail due to the MDR phenotype of the tumor cells. Additionally, agents augmenting immunologic effector function might provide clinical benefit in combination with SGN-33. The combination of SGN-33 and lenalidomide is now being evaluated in a clinical trial.

Pure Hematopoietic Stem Cells Reconstitute Immunity in Allogeneic Hosts Superior to Bone Marrow: Immunosuppression by Subclinical Graft-Versus-Host Disease.

Antigen specific immune responses are known to be impaired following allogeneic hematopoietic cell transplantation (HCT). Some clinical studies suggest that graft T cell depletion for the prevention of graft-versus-host disease (GVHD) leads to poorer immune recovery, while other correlate GVHD with delayed immune reconstitution. Our studies sought to examine the degree to which the co-transplantation of GVHD-inducing mature cells mediated protective immunity post-HCT. We compared the transplant of FACS purified hematopoietic stem cells (HSC: cKit+Thy1.1loSca+Lin-) with bone marrow (BM) between congenic (BA to B6Ly5.2), minor-antigen mismatched (BA to BALB.B), haploidentical (BAxSWR F1 to BALB/cxSWR F1) and MHC-mismatched (BA to BALB/c) donor and host pairs. We show that grafts composed solely of purified HSC give uniformly superior lymphoid reconstitution across all mismatched pairs, both qualitatively and quantitatively. Although absolute blood lymphocytes counts were increased in recipients of BM compared to HSC, lymphoid reconstitution as measured by lymph node size, counts and architecture was significantly improved in the HSC groups regardless of minor or major mismatches between donor and host. Proliferative responses to the allele specific peptides of the antigen hen egg lysozyme were also significantly increased in the HSC as compared BM recipients (p=0.028), with fully MHC mismatched BM recipient cells showing almost no proliferative response. The use of MHC allele specific antigens also revealed that T cell responses post-HCT are dominated by donor-restricted elements. These data suggest that subclinical GVHD mediated by mature cells in the donor BM result in impaired immune reconstitution. While there may be an increase in absolute lymphocyte numbers, this increase does not correlate with an increase in immune cell function. These findings provide important insight into the benefits of purified HSC for preventing post-HCT infectious complication in addition to its known GVHD prophylaxis benefits. Our data highlights the benefit of transplanting a pure HSC population, as the relatively small number of T cells that are found in mouse bone marrow or that remain in the graft after T cell depletion in humans can still result in immune impairment due to subclinical GVHD.

Effect of non-operative management (NOM) of splenic ruptureversussplenectomy on the distribution of peripheral blood lymphocyte populations and cytokine production by T cells

Post-traumatic splenectomy is associated with increased postoperative morbidity and mortality and long-term impairment of humoral and cellular immunity. Alternatives to surgery have been developed to minimize or avoid the immediate and/or long-term complications of splenectomy. Herein we investigated the long-term effect of non-operative management (NOM) of the traumatic rupture of the spleen on the distribution of peripheral blood (PB) lymphocyte populations and cytokine production by T cells. PB samples were drawn from six NOM patients, 13 age-matched adults who had undergone splenectomy after trauma (SP patients) and 31 age-matched controls. Cellular phenotypes and the intracellular production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-10 cytokines in T cells were determined in whole blood +/- mitogens by flow cytometry. NOM patients did not show any changes in the absolute numbers of lymphocytes or the distribution of their subsets, compared to the controls. In contrast, SP patients showed a sustained increase in the percentage and/or absolute numbers of lymphocytes, CD8 T cells, activated CD8 T cells, natural killer (NK) T cells, NK cells and gammadelta T cells, and a reduction in naive CD4 T cells. The constitutive or induced cytokine production by T cells of the NOM group was similar to the control group, whereas SP patients had increased percentages of constitutive IL-2- and IFN-gamma-producing CD8 T cells and IFN-gamma-producing CD4 T cells. Our findings indicate collectively that the healing process in NOM does not affect the architecture of the spleen to such an extent that it would lead to long-term alterations of the proportions of PB lymphocytes or the T cell cytokine profiles.

P156 Correlation of absolute lymphocyte numbers with prognosis in non-Hodgkins lymphoma patients

P156 Correlation of absolute lymphocyte numbers with prognosis in non-Hodgkins lymphoma patients

Prognostic nomogram and index for overall survival in previously untreated patients with chronic lymphocytic leukemia

AbstractThe clinical course for patients with chronic lymphocytic leukemia is extremely heterogeneous. The Rai and Binet staging systems have been used to risk-stratify patients; most patients present with early-stage disease. We evaluated a group of previously untreated patients with chronic lymphocytic leukemia (CLL) at initial presentation to University of Texas M. D. Anderson Cancer Center to identify independent characteristics that predict for overall survival. Clinical and routine laboratory characteristics for 1674 previously untreated patients who presented for evaluation of CLL from 1981 to 2004 were included. Univariate and multivariate analyses identified several patient characteristics at presentation that predicted for overall survival in previously untreated patients with CLL. A multivariate Cox proportional hazards model was developed, including the following independent characteristics: age, β-2 microglobulin, absolute lymphocyte count, sex, Rai stage, and number of involved lymph node groups. Inclusion of patients from a single institution and the proportion of patients younger than 65 years may limit this model. A weighted prognostic model, or nomogram, predictive for overall survival was constructed using these 6 characteristics for 5- and 10-year survival probability and estimated median survival time. This prognostic model may help patients and clinicians in clinical decision making as well as in clinical research and clinical trial design.

Systemic Immune Activation and Peripheral Blood Lymphocytes during Primary Chemotherapy with the Combination of Doxorubicin and Paclitaxel in Patients with Breast Carcinoma

Abstract In recent years, there has been interest in the study of alterations of the immune system of breast carcinoma patients undergoing primary chemotherapy. Changes in the phenotype, function of peripheral blood lymphocytes as well as alterations in the release of pro-inflammatory cytokines accompanying the administration of primary chemotherapy have been described. We have investigated urinary neopterin and peripheral blood leukocyte phenotype in 44 breast carcinoma patients treated with the combination of doxorubicin and paclitaxel. Compared to controls, patients with breast carcinoma had significantly lower relative number of CD3+ lymphocytes, and significantly higher relative and absolute numbers of natural killer cells, relative and absolute numbers of CD3+DR+ lymphocytes, relative and absolute numbers of CD3+CD69+ lymphocytes, relative numbers of CD8+CD57+ lymphocytes and CD14+DR+ monocytes and relative and absolute numbers of CD14+CD16+ monocytes. A significant correlation was observed before the initiation of therapy between urinary neopterin and the relative numbers of CD3+CD4+ lymphocytes (rs = - 0.411, p &lt; 0.01), absolute numbers of CD3+CD4+ lymphocytes (rs = - 0.313, p &lt; 0.05), relative number of CD3+CD8+ lymphocytes (rs = 0.352, p &lt; 0.05), CD4+/CD8+ ratio (rs = -0.404, p &lt; 0.01), relative numbers of CD3+DR+ lymphocytes (rs = 0.343, p &lt; 0.05), absolute numbers of CD3+DR+ lymphocytes (rs = 0.306, p &lt; 0.05), and relative numbers of CD8+CD57+ lymphocytes (rs = 0.410, p &lt; 0.01). Urinary neopterin and peripheral blood leukocyte phenotype was followed in 44 breast carcinoma patients treated by primary chemotherapy with doxorubicin and paclitaxel before the start of the treatment and on 3 subsequent visits in approximately monthly intervals. Compared to pretreatment values, urinary neopterin, relative and absolute numbers of CD3+, CD3+CD4+ and CD8+CD28+ lymphocytes were significantly increased throughout the course of treatment. No significant changes were observed in relative and absolute numbers of NK cells, CD8+- CD57+, CD3+DR+, CD3+CD25+, and CD3+CD69+ lymphocytes. In contrast, the relative and absolute numbers of CD19+ lymphocytes and CD19+CD23+ lymphocytes were significantly decreased throughout the course of therapy. The relative and absolute numbers of monocytes were significantly increased throughout the course of therapy. Significantly increased absolute numbers were also observed for CD14+DR+ and CD14+CD16+ monocytes. Among 37 evaluable patients, pathological complete response of the primary tumor, including regional lymph nodes, was observed in 6 cases. In patients with pathological complete response relative and absolute numbers of lymphocytes and absolute numbers of CD4+ lymphocytes were significantly lower at baseline. A total of 18 patients had evidence of pathological involvement of the lymph nodes. Compared to patients with lymph node involvement detected in surgical specimen, 19 patients without lymph node involvement had at baseline significantly lower relative and absolute number of lymphocytes, absolute numbers of CD3+, CD3+CD4+, CD3+DR+ and CD8+CD57+ lymphocytes. When ratio of peripheral blood leukocyte counts at visit 2/visit 1 was calculated, patients without lymph node involvement at surgery also had significantly increased ratio of relative lymphocyte numbers, relative and absolute CD14+DR+ monocyte numbers. In conclusion, systemic immune activation in breast carcinoma patients treated with doxorubicin and paclitaxel is associated with significant changes of peripheral blood leukocyte phenotype, including an increase of relative and absolute numbers of CD3+CD4+ lymphocytes.