Attrition of T-cell functions and simultaneous upregulation of inhibitory markers correspond with the waning of BCG-induced protection against tuberculosis in mice.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live attenuated vaccine. However, the correlates of protection and waning of its immunity against tuberculosis is poorly understood. In this study, we correlated the longitudinal changes in the magnitude and functional quality of CD4(+) and CD8(+) T-cell response over a period of two years after mucosal or parenteral BCG vaccination with the strength of protection against Mycobacterium tuberculosis in mice. The BCG vaccination-induced CD4(+) and CD8(+) T cells exhibited comparable response kinetics but distinct functional attributes in-terms of IFN-γ, IL-2 and TNF-α co-production and CD62L memory marker expression. Despite a near life-long BCG persistence and the induction of enduring CD4(+) T-cell responses characterized by IFN-γ and/or TNF-α production with comparable protection, the protective efficacy waned regardless of the route of vaccination. The progressive decline in the multifactorial functional abilities of CD4(+) and CD8(+) T cells in-terms of type-1 cytokine production, proliferation and cytolytic potential corresponded with the waning of protection against M. tuberculosis infection. In addition, simultaneous increase in the dysfunctional and terminally-differentiated T cells expressing CTLA-4, KLRG-1 and IL-10 during the contraction phase of BCG-induced response coincided with the loss of protection. Our results question the empirical development of BCG-booster vaccines and emphasize the pursuit of strategies that maintain superior T-cell functional capacity. Furthermore, our results underscore the importance of understanding the comprehensive functional dynamics of antigen-specific T-cell responses in addition to cytokine polyfunctionality in BCG-vaccinated hosts while optimizing novel vaccination strategies against tuberculosis.

Clinical impact of radiolabeled anti-CD4 antibodies in the diagnosis of rheumatoid arthritis.

Human rheumatoid arthritis (RA) is characterized by severe chronic synovitis with abundance of CD4-positive T-cells and macrophages in the inflamed synovial tissue. These cells likely play a central pathogenetic role in RA and experimental models of arthritis. CD4 is a surface molecule present on the helper/inducer subset of T lymphocytes and macrophages, although with a lower density on the latter. CD4+ T-cells/macrophages and their cytokine products, therefore, represent potential therapeutic and diagnostic targets in RA. CD4, a 55 kDa monomeric glycoprotein, binds as a T-cell coreceptor to conserved areas of the major histocompatibility complex II on antigen-presenting cells, and thereby participates in the formation of the immunological synapse and the provision of the so-called "second signal" required for full activation of T-helper cells. A specific diagnostic or therapeutic approach is the direct targeting of CD4+ T-cells by anti-CD4 monoclonal antibodies (mAbs). In addition to therapeutic clinical trials with anti-CD4 mAbs in RA, which have yielded only ambiguous results, anti-CD4 mAbs have also been developed and applied for diagnostic purposes. The studies thus far conducted in RA have focused on the following aspects: 1) comparison of anti-CD4 mAb imaging to the established early methylene diphosphonate (MDP) scan; 2) biodistribution/ pharmacokinetics studies; and 3) specificity of joint imaging with anti-CD4 mAbs in comparison to control immunoglobulins with irrelevant specificity. The available results in RA and arthritis models show that 99mTc-anti-CD4 mAbs are well-suited to actively image diseased joints, and clearly allow more specific imaging than 99mTc-MDP or control immunoglobulins. Because effective treatment is known to reduce the density of CD4+ cells in the inflamed synovial membrane, diagnostic methods targeted to CD4 warrant further attention, also for early diagnosis of clinically silent joints, precise description of the cellular infiltrates, and monitoring of anti-rheumatic therapy.

In vivo imaging of mucosal CD4+ T cells using single photon emission computed tomography in a murine model of colitis.

Immune responses that occur in the context of human infectious and inflammatory diseases are usually studied by sampling cells from peripheral blood, from biopsies, or by end-point harvests at necropsy. These approaches are likely to yield information that is incomplete and/or non-representative. Here, we report the development and validation of a non-invasive method to localize and to quantitate the disposition of specific subpopulations of cells in vivo. In a murine model of dextran sulfate sodium (DSS)-induced colitis, CD4+ T cells were visualized in the colon by single photon emission computed tomography (SPECT-CT) after injection of monoclonal, non-depleting, indium-111 (111In) labeled anti-CD4+ antibodies. The SPECT-CT colon uptake ratio (CUR) was found to correlate (p<0.01) with the number of total CD4+ T cells and with standard measures of pathology (colon length, cell counts, and histopathologic evidence of apoptosis, edema, and cellular infiltrates) as assessed by direct examination of diseased colon. Each of these parameters, including the SPECT-CT signal uptake, increased as a function of DSS dose (p<0.05). We conclude that CT-SPECT imaging using an 111In-labeled anti-CD4+ antibody is reflective of traditional parameters of pathology in this experimental model of murine colitis. This approach should be readily applicable to the imaging of discrete cell subpopulations in non-human primates and in humans, thus augmenting our understanding of infectious diseases and inflammation in vivo.

In vivo evaluation of 111In-labeled T-lymphocyte homing in experimental colitis.

UNLABELLED: Blockade of lymphocyte recruitment to the intestinal mucosa is considered a useful therapy for inflammatory bowel disease (IBD) and anti-alpha4 antibodies have clinical benefit in patients with active Crohn's disease. The aim of this study was to evaluate a scintigraphic technique to assess lymphocyte homing to the colon in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis (TNBS colitis) in vivo. METHODS: TNBS-sensitized and nonsensitized murine total lymphocytes or CD4+ lymphocytes were radiolabeled with 111In-oxinate. Cells were injected into control mice (n = 5) or mice with TNBS colitis (n = 5). Specific abdominal radioactive uptake was determined by SPECT using a dedicated pinhole system 48 h after cell transfer. Radioactive colon uptake was correlated with histology and colon weight as parameters of inflammation. RESULTS: The radioactive colon uptake was most evident in mice with TNBS colitis that received sensitized lymphocytes (uptake ratio [mean +/- SEM], 0.51 +/- 0.03 vs. 0.22 +/- 0.04; P = 0.004). The sensitized 111In-labeled lymphocytes exacerbated colitis compared with nonsensitized lymphocytes. The colon uptake correlated well with both colon weight and histologic score (R2 = 0.836 and 0.933, respectively). The use of purified 111In labeled CD4+ lymphocytes resulted in a similar scintigraphic pattern. Administration of an anti-alpha4 antibody decreased radioactivity colon uptake of the (111)In-labeled cells compared with the control antibody in mice with TNBS colitis (uptake ratio, 0.72 +/- 0.14 to 0.33 +/- 0.03; P = 0.012). CONCLUSION: Animal pinhole SPECT can be applied for temporal and spatial analysis of the lymphocyte homing process in experimental colitis. This technique makes possible the in vivo evaluation of therapeutic efficacy of new drugs that interfere with lymphocyte migration. Moreover, colon uptake of radioactivity can be used as a parameter of disease activity in experimental colitis.

Technetium-99m scintigraphy to visualize T-cell homing in vivo: a preclinical study.

The knowledge of lymphocyte distribution is very usefulness in monitoring therapeutic treatments. We present here a method employed in clinical practice, the scintigraphy, to study in the rat the physiologic lymphocyte traffic. Rat T cells labeled with 99mTc were injected in syngeneic animals, and their fate was studied by serial scintigraphic scanning. Sorted naïve CD4+ CD45RC(bright) T cells homed to lymphoid organs and accumulated in spleen. CD4+ CD45RC(dim) memory lymphocytes first reached the liver and the lungs and recirculated. The results obtained by using the scintigraphic method to in vivo study the lymphocyte homing in rats are comparable to those obtained with previously used experimental methods. We consider the scintigraphic method a useful tool to in vivo track lymphocytes and to address therapeutic treatment in men.

In vivo tissue distribution of CD4 lymphocytes in mice determined by radioimmunoscintigraphy with an 111In-labeled anti-CD4 monoclonal antibody.

The tissue distribution of CD4 lymphocytes in normal C57/BL mice and CD4 knockout mice was determined by biodistribution measurements and gamma camera imaging with an 111In-labeled rat IgG2b monoclonal antibody directed against the murine CD-4 antigen. In normal mice high concentrations of antibody accumulated in the spleen and lymph nodes. At 45 hr after injection, the concentration of radiolabel in the spleen and lymph nodes of normal mice were 10- to 20-fold greater than in the corresponding tissue of the CD4 knockout mice and nonlymphoid tissues of both types of mice. At 24 and 45 hr, gamma camera images showed high concentrations of radiolabeled antibody in lymph node and spleen of normal but not knockout mice. These results indicate that radioimmunoscintigraphy with 111In-anti-CD4 is an excellent method for studying tissue distribution of CD lymphocytes in mice. Using an equivalent anti-human CD antibody, this method might be useful for studying the pathophysiology of conditions in which these cells play a critical role and for monitoring therapies for these disorders.