CD8 Lymphocyte Populations Research Articles

Single-cell profiling reveals distinct immune response landscapes in tuberculous pleural effusion and non-TPE.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and remains a major health threat worldwide. However, a detailed understanding of the immune cells and inflammatory mediators in Mtb-infected tissues is still lacking. Tuberculous pleural effusion (TPE), which is characterized by an influx of immune cells to the pleural space, is thus a suitable platform for dissecting complex tissue responses to Mtb infection. We employed singe-cell RNA sequencing to 10 pleural fluid (PF) samples from 6 patients with TPE and 4 non-TPEs including 2 samples from patients with TSPE (transudative pleural effusion) and 2 samples with MPE (malignant pleural effusion). Compared to TSPE and MPE, TPE displayed obvious difference in the abundance of major cell types (e.g., NK, CD4+T, Macrophages), which showed notable associations with disease type. Further analyses revealed that the CD4 lymphocyte population in TPE favored a Th1 and Th17 response. Tumor necrosis factors (TNF)-, and XIAP related factor 1 (XAF1)-pathways induced T cell apoptosis in patients with TPE. Immune exhaustion in NK cells was an important feature in TPE. Myeloid cells in TPE displayed stronger functional capacity for phagocytosis, antigen presentation and IFN-γ response, than TSPE and MPE. Systemic elevation of inflammatory response genes and pro-inflammatory cytokines were mainly driven by macrophages in patients with TPE. We provide a tissue immune landscape of PF immune cells, and revealed a distinct local immune response in TPE and non-TPE (TSPE and MPE). These findings will improve our understanding of local TB immunopathogenesis and provide potential targets for TB therapy.

RELATIONSHIP OF LYMPHOCYTE SUBPOPULATIONS IN BREAST CANCER PATIENTS WITH TREATMENT RESULTS

Introduction. Breast cancer (BC) is an immunogenic tumor. Immune cells infiltration of tumor tissue can affect the clinical course of the disease. The immunogenicity of breast cancer varies depending on the molecular subtype.The aim of this work was to study the main indicators of systemic and local immunity before patient’s treatment and to determine their relationship with the immediate neoadjuvant chemotherapy results.Materials and methods. Patients with stage II–III BC received standard neoadjuvant chemotherapy in accordance with the molecular subtypes. The percentage of the main effector and regulatory lymphocytes populations of systemic and local immunity was determined by flow cytometry.Results. A decrease in the level of effector CD8 and CD4 lymphocyte populations and an increase in the level suppressor populations in tumor tissue in comparison with peripheral blood indicate an immunosuppressive state of local immunity in BC patients. In tumor tissue, a high level of CD8+ PD-1+ and CD4+ PD-1+ cells were associated with a high level of regulatory CD4+ CD25highCD127–/low and CD8+ CD11b– CD28– lymphocytes. Differences were found in the significance of individual lymphocyte populations for the immediate results of treatment between patients with different subtypes of breast cancer.Conclusion. Determination of lymphocyte subpopulations correlating with the level of PD-1 cells, and the results of treatment in patients with different molecular BC subtypes, will help a clearer understanding of the antitumor immune response in this pathology, and will also serve as a basis for identifying immune biomarkers that can be used as additional predictive factors in various treatment options for BC patients.

Effect of β-Mannanase Supplementation in Conventional Diets on the Performance, Immune Competence and Intestinal Quality of Broilers Challenged With Eimeria sp.

The objective of this work was to study the effect of β-mannanase supplementation in diets of broilers challenged with Eimeria sp. on performance, intestinal morphology, and immune profile. A total of 320 male chicks were housed in a completely randomized design in 2 × 2 factorial scheme (with and without β-mannanase and with and without challenge with coccidiosis vaccine at 14 d of age). Feed intake, a week after the experimental challenge, was higher (P < 0.05) in birds that did not receive the enzyme supplementation. During the period from 1 to 21 d of age, the live weight and weight gain were higher (P < 0.05) for birds that were not supplemented. From 1 to 42 d, supplementation with β-mannanase resulted in worst feed conversion. One week after the challenge, the birds that consumed control diets and that were challenged had higher (P < 0.05) proportion of activated T CD8 lymphocyte populations. Unchallenged birds supplemented with the enzyme exhibit higher proportions of CD4. At this same age, there was greater (P < 0.05) crypt depth, smaller (P < 0.05) villus: crypt proportion, and higher (P < 0.05) number of goblet cells in the intestinal mucosa of the challenged birds compared to the control. However, at the age of 42 d, the birds that received the enzyme supplementation showed a lower crypt depth (P < 0.05), which resulted in a better villus: crypt proportion. Although not improving the performance of birds, supplementation with β-mannanase contributed positively to the quality and intestinal health of the broilers.

Abstract 2546: Impact of CDK4/6 inhibition on tumor immune microenvironment and immunomodulatory effects PD-L1 blockade in a physiologically relevant 3D-tumoroid model of fresh patient tumor tissue ex vivo

Abstract Introduction: In mammalian cells, exposure to growth factors triggers de novo synthesis of D-type cyclins, which then associate with their catalytic partners, cyclin dependent kinase CDK4 or CDK6 to regulate the cell cycle. Recent studies implied the role of CK4/6 inhibitors such as Abemaciclib in modulating tumor immune microenvironment and response to PD1/PD-L1 inhibitors. However, the mechanisms by which CD4/6 inhibitors affect the tumor microenvironment is not well understood. Here we investigated the effect of CDK4/6 inhibition by Abemaciclib on the tumor immune microenvironment and response to PD1/PD-L1 inhibitors using Nilogen’s 3D-EX ex vivo drug screening platform utilizing un-propagated 3D tumoroids of fresh patient tumor tissue with an intact tumor microenvironment. Materials and Methods: All non-small cell lung carcinoma tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D tumoroids measuring 100-150 micron in size were treated with Abemaciclib, pembrolizumab (Keytruda) alone or in combination. Treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages and cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Multiplex cytokine assays were performed to assess drug-induced changes in cytokine release. High-content confocal analysis was used to quantify tumor cell killing. Results: We showed that drug treatments had significant impact on immune cell compositions, specifically on M1/M2 macrophage polarization as well as on CD4 and CD8 lymphocyte populations in selected tumors, which was accompanied by significant changes in pro-inflammatory cytokine release. We further characterized pharmacological interaction between Abemaciclib and pembrolizumab and correlated with tumor cell killing in quantitative imaging studies. Conclusion: In this comprehensive study we documented the pharmacological effect of CDK4/6 inhibition on tumor immune microenvironment in a physiologically relevant 3D tumoroid model of ex vivo drug testing. We further characterized the pharmacological interaction between Abemaciclib and pembrolizumab to better understand the mechanisms by which CDK4/6 inhibition may enhance therapeutic response to PD1 blockade in patient tumors. Citation Format: Melba Marie Page, Melanie Mediavilla Varela, Vijayendra Agrawal, Jenny Kreahling, Soner Altiok. Impact of CDK4/6 inhibition on tumor immune microenvironment and immunomodulatory effects PD-L1 blockade in a physiologically relevant 3D-tumoroid model of fresh patient tumor tissue ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2546.

Response to Intravenous Allogeneic Equine Cord Blood-Derived Mesenchymal Stromal Cells Administered from Chilled or Frozen State in Serum and Protein-Free Media.

Equine mesenchymal stromal cells (MSC) are commonly transported, chilled or frozen, to veterinary clinics. These MSC must remain viable and minimally affected by culture, transport, or injection processes. The safety of two carrier solutions developed for optimal viability and excipient use were evaluated in ponies, with and without allogeneic cord blood-derived (CB) MSC. We hypothesized that neither the carrier solutions nor CB-MSC would elicit measurable changes in clinical, hematological, or biochemical parameters. In nine ponies (study 1), a bolus of HypoThermosol® FRS (HTS-FRS), CryoStor® CS10 (CS10), or saline was injected IV (n = 3/treatment). Study 2, following a 1-week washout period, 5 × 107 pooled allogeneic CB-MSCs were administered IV in HTS-FRS following 24 h simulated chilled transport. Study 3, following another 1-week washout period 5 × 107 pooled allogeneic CB-MSCs were administered IV in CS10 immediately after thawing. Nine ponies received CB-MSCs in study 2 and 3, and three ponies received the cell carrier media without cells. CB-MSCs were pooled in equal numbers from five unrelated donors. In all studies, ponies were monitored with physical examination, and blood collection for 7 days following injection. CD4 and CD8 lymphocyte populations were also evaluated in each blood sample. In all three studies, physical exam, complete blood cell count, serum biochemistry, and coagulation panel did not deviate from established normal ranges. Proportions of CD4+ and CD8+ lymphocytes increased at 168 h postinjection in CB-MSC treatment groups regardless of the carrier solution. Decreases in CD4+/CD8+ double positive populations were observed at 24 and 72 h in CB-MSC-treated animals. There was no difference in viability between CB-MSCs suspended in HTS-FRS and CS10. HTS-FRS and CS10 used for low volume excipient injection of MSC suspensions were not associated with short-term adverse reactions. HTS-FRS and CS10 both adequately maintain CB-MSC viability following hypothermic or frozen simulated transport, respectively. CB-MSCs do not elicit clinical abnormalities, but allogeneic stimulation of CD4+ and CD8+ lymphocyte populations may occur. Future studies should include in vitro or in vivo evaluation of cell-mediated or adaptive immunity to autologous, identical allogeneic, or MSC originating from additional unrelated individuals in order to better characterize this response.

NLRP12 protein function in the regulation of MHC-I expression in monocytes of patients transplanted with HPCs and their relationship to the reconstitution of CD8 T lymphocytes. (P1333)

Abstract The reconstitution of the T CD8 lymphocyte population in patients subject to hematopoietic cell transplantation (HCT) depends largely of homeostatic peripheral expansion (HPE) of the T CD8 lymphocytes that were not eliminated during the pre-transplant conditioning regimen or the ones that are contained in the transplanted cells. In order to have an efficient HPE, MHC-I molecules, IL-7 and IL-15 are required. Evidences obtained in our lab indicate that the low and/or heterogeneous MHC-I levels of expression correlate with a inefficient reconstitution of T CD8 lymphocytes in patients that received allogeneic HCT. The deficiency in MHC-I expression could be due to a decrease in the expression of the NLRP12 protein, which has been proposed as a positive regulator of classical and non-classical MHC-I molecules expression. Therefore the objective of the following project is to determine the involvement of NALP12 in the MHC-I expression in monocytes from patients subject to HCT. We studied a cohort of 11 patients, 6 of which received autologous transplant and 5 with allogeneic transplant. Most of the patients present normal levels of T CD8 lymphocytes and a homogeneous expression of MHC-I. The patients that received autologous transplantation show a correlation of r=0.56. Our results suggest that NALP12 could act as an enhancer for the expression of the MHC-I molecules and that this expression could be affected in patients subject to allogeneic HPC transplantation.

Analysis of Clinical Outcome and Survival in Pediatric Patients Undergoing Extracorporeal Photopheresis for the Treatment of Steroid-refractory GVHD

Extracorporeal photopheresis (ECP) has been used for treatment of steroid-refractory graft-versus-host disease (GVHD) with encouraging results. However, only a few pediatric centers have experienced with ECP, mainly by technical difficulties of leukapheresis in children. We retrospectively analyzed 27 patients treated during 5 years with ECP for steroid-refractory acute and chronic GVHD. We performed an "off-line" approach using a continuous flow cell separator (Cobe Spectra) and a Ultraviolet A irradiator (UVAMATIC). Among acute GVHD patients, 11 of 21 obtained a complete remission (CR) and another 8 reached partial remission. In the chronic GVHD group, 3 patients obtained CR and another 2 were in partial remission. Disease-free survival probability was 43±9% in the whole group. The only variable that impacts on disease-free survival on multivariate analysis was achieving CR with ECP. We also found a better CD4/CD8 ratio after ECP. In detail, among CD4 lymphocyte population, an increase in the effector memory and T-regulatory population was found. We may conclude that ECP is a safe and effective steroid-refractory GVHD treatment using the ex-vivo collection method, even in small children. Clinical response favorably impacts on long-term survival and seems to be associated with peripheral blood lymphocyte subset changes generating peripheral tolerance.

INITIATION OF HIV THERAPY

In this paper, we numerically show that the dynamics of the HIV system is sensitive to both the initial condition and the system parameters. These phenomena imply that the system is chaotic and exhibits a bifurcation behavior. To control the system, we propose to initiate an HIV therapy based on both the concentration of the HIV-1 viral load and the ratio of the CD4 lymphocyte population to the CD8 lymphocyte population. If the concentration of the HIV-1 viral load is higher than a threshold, then the first type of therapy will be applied. If the concentration of the HIV-1 viral load is lower than or equal to the threshold and the ratio of the CD4 lymphocyte population to the CD8 lymphocyte population is greater than another threshold, then the second type of therapy will be applied. Otherwise, no therapy will be applied. The advantages of the proposed control strategy are that the therapy can be stopped under certain conditions, while the state variables of the overall system is asymptotically stable with fast convergent rate, the concentration of the controlled HIV-1 viral load is monotonic decreasing, as well as the positivity constraint of the system states and that of the dose concentration is guaranteed to be satisfied. Computer numerical simulation results are presented for an illustration.

CD8+CD28− cells and CD4+CD25+ regulatory T cells in the peripheral blood of advanced stage lung cancer patients

To evaluate the CD8+CD28- and CD4+CD25+ regulatory T (Treg) cells in addition to other some lymphocyte subgroups in peripheral blood of advanced stage lung cancer patients. The study group (n = 28) comprised chemotherapy and radiotherapy naïve patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The control group (n = 22) consisted of age- and sex-matched healthy volunteers. Flow cytometry was used to count T cells, natural killer (NK) cells and CD4+CD25 Treg cells, and for CD8+ T cell subgroup analysis. Flow cytometry was performed and annexin V binding was used for apoptotic cell evaluation. In patient group, the percentage of CD8+CD28- cells among lymphocytes was elevated, and there was also an increase in the CD28-/CD28+ cell ratio among CD8 lymphocyte population. The distribution of CD8 cells was different in lung cancer patients when compared with the control group. The absolute count of CD4+CD25(bright) cells and the percentages of these cells among total lymphocytes were higher in the patient group. The Annexin V(+) cell percentages among CD8+CD28- and CD8+CD28+ lymphocytes were higher in the patient group than in the control group. No differences were found between the NSCLC and SCLC patients with respect to the hematological parameters and the distribution of lymphocyte subgroups. In NSCLC patients, the percentage of CD8+CD28- cells among the lymphocyte population was higher in patients with stage IV than those with stage III. These findings may reflect the possibility of tumor-induced immunosuppression and they should be complemented with further studies.

HLA-DR Expressing Peripheral T Regulatory Cells in Newly Diagnosed Patients with Different Forms of Autoimmune Thyroid Disease

Several reports have claimed a role for T regulatory cells (Tregs) in the pathogenesis of various autoimmune diseases, including autoimmune thyroid disease (AITD). The aim of the present study was to examine whether changes in the number of peripheral CD4 + CD25highHLA-DR + lymphocytes, a subpopulation of Tregs, occur in patients with AITD. Three-color flow cytometry was used to detect the proportion of CD4 cells expressing CD25, CD25high, and HLA-DR in 70 newly diagnosed and untreated AITD patients and 20 controls. The intensity of CD25 expression on these cells was also examined. The proportion of CD4 + CD25 + cells as well as the proportion of CD4 + CD25high cells among the population of CD4 lymphocytes was not different in AITD patients relative to controls. However, a significant increase in the proportion of CD4 + CD25highHLA-DR + cells among the population of CD4 lymphocytes was found in patients with Hashimoto's thyroiditis (HT) compared to controls. In HT patients there is a quantitative increase of CD4 + CD25highHLA-DR + cells that may indicate a compensatory expansion of this subpopulation of Tregs in an attempt to suppress the immune response.

CD4&lt;sup&gt;+&lt;/sup&gt; lymphocyte reference ranges in healthy adults in Port Harcourt, Nigeria

Aims and Objectives : Reference values are essential for the interpretation of data in clinical practice and research studies. The aim of this study was to establish reference ranges for CD4 lymphocytes in healthy HIV-negative Nigerians. Methodology : 200 healthy Human Immunodeficiency Virus (HIV) antibody free individuals constituted the subjects for this study. 150 HIV-infected without AIDS-defining illness and 150 HIV-infected with AIDS-defining illness were included as controls. Results : Participants were aged 18-63years (mean age 34 ± 9.2years). the CD4 lymphocyte count of healthy Nigerians mean SD 685 ± 99, 95% CI 487 883 was twice that of HIV-infected subjects without AIDS-defining illness (314 ± 45, 95% CI 224 404). The CD4 lymphocyte count of HIV-infected with AIDS-defining illness was one fourth or less (148 ± 30, 95% CI 88 208). The mean AIDS-defining CD4 lymphocyte count was (148 30 cells/ul). Healthy women had significantly higher mean absolute CD4 lymphocyte count (729 ± 87, 95% CI 555 to 903) compared to men (647 ± 93, 95% CI 460 to 833) (p Conclusion : Caucasian CD4 lymphocyte reference values are not applicable to Nigerians, as it does not reflect the Nigerian CD4 lymphocyte population. Keywords : CD4 lymphocyte count, reference ranges, Port Harcourt, Nigerians Sahel Medical Journal Vol. 8(3) 2005: 71-75

Immune Function Assay Enters the Clinical Laboratory

Methods for assessing a patient’s state of immune responsiveness has traditionally involved the use of radioactive markers to purified populations of lymphocytes after they have been stimulated. Briefly, a lymphocyte stimulant or mitogen such as phytohemagglutinin (PHA) is added to isolated lymphocytes. Lymphocytes in the peripheral blood express receptors on the cell surface, which bind to mitogens or specific antigens presented in conjunction with the major histocompatibility complex (MHC). Exposure to the mitogen or antigen results in activation and expansion of the lymphocyte subpopulation reactive to the antigen or mitogen. The suspension can then be labeled with a radioactive marker such as 3 H thymidine where DNA synthesis is measured on a scintillation counter. The drawbacks of this assay include a long turnaround time (72 hours) and the use of radioactive materials. A new assay, Immuknow by Cylex (Columbia, MD) was FDA approved in 2002 for measuring global cell mediated immunity. Heparinized whole blood is diluted in sample diluent and stimulated with the mitogen PHA. After an overnight incubation period at 37°C in 5% CO 2 , the CD4 lymphocyte population is isolated using a magnetic separation device and monoclonal antibodies to CD4. After a series of washes, the target cell population is lysed releasing cellular adenosine triphosphate (ATP), which is measured using a microplate luminometer and the luciferase-luciferin enzyme system. In the presence of luciferin and Mg-ATP, luciferase catalyzes a reaction producing oxyluciferin and light which is directly proportional to the ATP concentration 1 in the patient sample. An unstimulated control of patient sample is run in parallel, and the results are expressed as the change or delta between the stimulated and non-stimulated wells. A standard curve utilizing sequential ATP concentrations is incorporated in the test system. This assay has a 24-hour turnaround time, utilizes whole blood, and requires no radioactive materials.

P‐9 CD8 and CD4 lymphocyte populations in blood from healthy dogs and dogs naturally infected with Ehrlichia canis

The purpose of this study was to assess the presence of an immune dysregulation in dogs naturally infected with Ehrlichia canis. We compared CD4 and CD8 lymphocyte populations in a group of dogs with ehrlichiosis and a group of healthy dogs. Thirty‐seven dogs with natural canine ehrlichiosis (Group A) and 15 unaffected dogs (Group B) were included in the study. The study of lymphocyte populations was made by flow cytometry. Monoclonal antibodies against CD3, CD4 and CD8 were used. Results obtained in this study show an inversion in the CD4:CD8 ratio in the blood of dogs with ehrlichiosis when compared with the CD4:CD8 ratio in blood from control dogs. In healthy dogs, the number of CD4 T lymphocytes was greater than CD8 T lymphocytes. However, in dogs with ehrlichiosis, the number of CD8 T lymphocytes was greater than CD4 T lymphocytes. The percentage of CD4 T lymphocytes was lower in dogs with ehrlichiosis than in healthy dogs, but significant differences were not found between both groups. However, mean value of the percentage of CD8 T lymphocytes (with respect to lymphocyte population) in dogs with ehrlichiosis (45.81 ± 20.90%) was significantly higher (P = 0.001) than in healthy dogs (25.41 ± 13.63%). These results confirm the presence of an alteration in the T lymphocyte subpopulations of dogs with ehrlichiosis, suggesting the presence of an immune dysregulation. Funding: Self‐funded.

Alterations in blood leucocyte adhesion molecule profiles in HIV-1 infection.

CD4 and CD8 lymphocyte numbers in the gut lamina propria are grossly altered in HIV-1 infection, out of proportion to alterations in the circulation. Such alterations in lymphocyte counts in the tissues may be due to altered leucocyte migration from the blood. One factor affecting leucocyte migration is adhesion molecule expression. Levels of adhesion molecule expression on peripheral CD4 and CD8 lymphocytes, monocytes and neutrophils from HIV-1-infected (AIDS and non-AIDS) and low-risk control individuals were compared. CD11a, CD62L, CD44, CD49d and beta7 integrin expression were examined by FACS analysis of fresh whole blood. Significant alterations in adhesion molecule expression were detected in HIV infection. The most striking alterations were observed in the CD8 lymphocyte population. CD11a expression was increased and CD62L and CD44 decreased. The CD4 lymphocyte population followed a similar, though less striking, pattern of alteration in adhesion molecule expression. Neutrophils displayed significantly reduced expression of both CD11a and CD62L, but only after onset of AIDS. Monocytes from infected individuals without AIDS displayed a different pattern of altered adhesion molecule expression compared with individuals with AIDS. These findings suggest that in HIV infection, leucocyte functions, such as migration, which require adhesion molecules are abnormal.

Lung-specific expression of adenovirus E3-14.7K in transgenic mice attenuates adenoviral vector-mediated lung inflammation and enhances transgene expression.

Herein, we report that the adenovirus E3-14.7K protein inhibits the inflammatory response to adenovirus in transgenic mice in which the E3-14.7K gene was selectively expressed in the respiratory epithelium, using the human surfactant protein C (SP-C) promoter. E3-14.7K mRNA and protein were detected specifically in the lungs of SPC/E3-14.7K transgenic mice. Responses of the transgenic mice to Av1Luc1, an E1-E3-deleted Ad vector encoding the luciferase reporter gene, were examined, including vector transgene expression and lung inflammation. In wild-type mice, luciferase activity declined rapidly and was lost 14 days following Av1Luc1 administration. The loss of luciferase activity was associated with pulmonary infiltration by macrophages and lymphocytes. In heterozygous SPC/E3-14.7K mice, luciferase activity was increased by 7 days compared with control littermates, and pulmonary infiltration by macrophages was decreased. In homozygous (+/+) SPC/E3-14.7K mice, luciferase activity was increased 7, 14, and 21 days following administration compared with wild-type mice, and lung inflammation was markedly reduced. After Av1Luc1 administration, PCNA staining of bronchiolar and alveolar respiratory epithelial cells was decreased in SPC/E3-14.7K transgenic mice, indicating decreased epithelial cell proliferation, a finding consistent with the observed reduction in inflammation. CD4 and CD8 lymphocyte populations were only mildly altered, while humoral responses to adenoviral vectors were unchanged in the SPC/E3-14.7K mice. The E3-14.7K protein expressed selectively in respiratory epithelial cells suppresses Ad-induced pulmonary epithelial cell cytotoxicity and lung inflammation in vivo and prolongs reporter gene expression.

Differential expression of cytokine genes in CD27-positive and -negative CD4 lymphocyte subsets from healthy humans and rheumatoid arthritis patients.

Immunofluorescence analysis of CD27 expression by CD4 lymphocytes from the peripheral blood of healthy humans or rheumatoid arthritis (RA) patients and from the synovial fluid (SF) of RA patients was carried out, along with the estimation of cytokine gene [interleukin (IL) 2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-10 and interferon-gamma (IFN-gamma)] expression in these lymphocyte subsets by RT-PCR. Although no differences in CD27-positive and -negative peripheral blood CD4 cell subset distribution were revealed, marked differences in IL-3, IL-4, IL-5 and IFN-gamma mRNA expression were detected between these lymphocyte subsets and between control and disease states. These results showed that phenotyping of different cell subsets in disease cannot provide adequate information about lymphocyte functional status. To estimate differences in cytokine gene expression, CD4 lymphocytes from the peripheral blood and SF of RA patients were compared. In both cases, mRNAs for IL-2, IL-4, IL-10 and IFN-gamma were detected, but CD4 cells from SF failed to express detectable levels of IL-5 mRNA despite our findings of a CD27-cell accumulation within the synovial population of CD4 lymphocytes. These are the first data to demonstrate that expression of the IL-5 gene in RA SF CD27- lymphocytes is down-regulated and that IL-5 disregulation in RA cannot be ruled out.

CD4 Lymphocytes in Women with Invasive and Preinvasive Cervical Neoplasia

Objective.To assess the relationship between CD4 lymphocyte population and stage of disease in cervical neoplasia.Methods.Study population was 107 women with invasive cervical cancer, 116 women with cervical intraepithelial neoplasia (CIN), and 32 women without neoplasia diagnosed in 1988–1994. All women under age 50 were seronegative for the human immunodeficiency virus (HIV). All women over age 50 with CD4:CD8 ratio below normal were HIV-negative. Stage was defined by FIGO criteria using clinical findings. CD4 and CD8 lymphocyte populations were enumerated by flow cytometry prior to treatment. The normal range of CD4 counts was defined as 537–1571 cells/mm3.Results.Distribution of CD4 count was similar in stages I (n= 40), II (n= 24), and III (n= 32), with 31% below normal and 9% above normal (mean CD4 count = 881). However, in stage IV (n= 11), 64% were below normal and 18% above normal (mean CD4 = 591). The difference in distribution between stages I–III and stage IV was statistically significant. Among 116 CIN patients, 10% had CD4 counts below normal and 3% above normal (mean CD4 = 910). Among 32 women without cervical neoplasia, 0% had CD4 counts below normal and 3% above normal. The difference between CIN and invasive cancer in the distribution of CD4 counts and CD8 counts was significant (P< 0.01). There was no difference in the CD4 count distribution by CIN severity. Forty-five percent of patients with below-normal CD4 counts at diagnosis developed recurrent cancer compared to 43% of patients with normal or above-normal CD4 counts.Conclusion.Women with invasive cervical cancer have lower CD4 counts and a broader distribution compared to women with preinvasive or no neoplasia. Metastatic cancer at diagnosis was associated with severely depressed CD4 count.

Relationship of transforming growth factor beta 1 to extracellular matrix and stromal infiltrates in invasive breast carcinoma.

Transforming growth factor beta (TGF-beta) comprises a group of multifunctional regulatory proteins, whose effects include stimulation of extracellular matrix formation and modification of immune function. The presence of TGF-beta 1 and TGF-beta 2 in invasive breast carcinomas has been determined and related to pathological features, the presence of fibronectin and tenascin and lymphocyte/macrophage infiltration, using immunohistochemistry. Differences were observed in the extent of reactivity within the same carcinoma and between tumours stained with an antibody detecting TGF-beta 1 ane one detecting TGF-beta plus TGF-beta 2, the latter having a higher level of reactivity. Prominent reactivity for TGF-beta 1 was associated with lymph node metastasis, (0.02 > P > 0.01), increased detection of cellular fibronectin, fine stromal fibronectin staining, more prominent reactivity for tenascin (0.02 > P > 0.01), the presence of tumour-associated macrophage infiltration and altered ratios of CD4 and CD8 lymphocyte populations, with CD8 lymphocytes predominating. These associations were not observed for carcinomas showing prominent staining with antibody detecting TGF-beta 2 as well as TGF-beta 1. The findings indicate that TGF-beta 1 may have a role in invasion and metastasis of breast carcinomas.

Viral‐Specific Immunization in AIDS‐Related Complex by Photopheresis

The potential for therapeutic intervention in 7 patients with AIDS-related complex (ARC) was evaluated through the use of photopheresis. The rationale for the study was based on: 1. the demonstration that psoralen and UVA could inactivate HIV/virus in vitro; 2. CD4 cells are the primary target population effected by HIV and photopheresis; and 3. reinfusion of inactivated virus and cell-associated virus might serve to engender an immune response. Preliminary results in 7 patients with ARC over 6 to 18 months revealed a virus-specific response with an elevation of HIV antibodies, while EBV and CMV titers remained unchanged. The immunologic results revealed an increase in the CD8 lymphocyte population, stable activation markers (B2 microglobulin neopterin), a decrease in p24 antigen titers and inability to culture HIV virus in 3 patients. All of these results were in the context of a stable or increasing CD4+ percent. Six patients did not reveal a generalized inhibition of other immune responses as demonstrated by recovery of DTH. In addition, the resolution of lymphadenopathy, night sweats, fever and weight loss, paralleled the immunologic response.

Phase 1 study of recombinant human CD4-immunoglobulin G therapy of patients with AIDS and AIDS-related complex

The safety and pharmacokinetics of recombinant CD4-immunoglobulin G (rCD4-IgG) were evaluated in a phase 1 study with dose escalation. A total of 16 patients, 6 with AIDS and 10 with AIDS-related complex, were evaluated at two university-affiliated hospital clinics. rCD4-IgG was administered once weekly for 12 weeks to four patients each at doses of 0.03, 0.1, 0.3, and 1.0 mg/kg of body weight. Dosing was intravenous for two patients in the 1.0-mg/kg dose group and intramuscular for the remaining patients. Dosing was intravenous for two patients in the 1.0-mg/kg dose group and intramuscular for the remaining patients. Pharmacokinetic, toxicity, and immunologic variables were monitored with all patients. Administration of rCD4-IgG was well tolerated, with no important clinical or immunologic toxicities noted. No subjects required dose reduction or discontinuation of therapy due to toxicity. No consistent changes were seen in human immunodeficiency virus antigen levels in serum or CD4 lymphocyte populations. The volume of distribution was small, and compared with that of rCD4, the half-life of the hybrid molecule was markedly prolonged following intramuscular or intravenous administration. The rate and extent of absorption following intramuscular dosing were variable. Intramuscular administration of rCD4-IgG appears to be inferior to intravenous dosing from a pharmacokinetic standpoint, with lower peak concentrations and variable absorption. After intravenous administration, peak concentrations of rCD4-IgG in serum (20 to 24 micrograms/ml) that have shown antiviral activity in vitro against more sensitive clinical isolates of human immunodeficiency virus were achieved. The peak concentrations in serum after intramuscular administration were below these levels. Treatment with rCD4-IgG was well tolerated at the doses administered to patients in this study but did not result in significant changes in CD4 lymphocyte counts or p24 antigen levels in serum.