Limit Theorems for Hybridization Reactions on Oligonucleotide Microarrays

We derive herein the limiting laws for certain stationary distributions of birth-and-death processes related to the classical model of chemical adsorption-desorption reactions due to Langmuir. The model has been recently considered in the context of a hybridization reaction on an oligonucleotide DNA microarray. Our results imply that the truncated gamma- and beta- type distributions can be used as approximations to the observed distributions of the fluorescence readings of the oligo-probes on a microarray. These findings might be useful in developing new model-based, probe-specific methods of extracting target concentrations from array fluorescence readings

Intratumoral Convergence of the TCR Repertoires of Effector and Foxp3+ CD4+ T cells

The presence of Foxp3+ regulatory CD4+ T cells in tumor lesions is considered one of the major causes of ineffective immune response in cancer. It is not clear whether intratumoral Treg cells represent Treg cells pre-existing in healthy mice, or arise from tumor-specific effector CD4+ T cells and thus representing adaptive Treg cells. The generation of Treg population in tumors could be further complicated by recent evidence showing that both in humans and mice the peripheral population of Treg cells is heterogenous and consists of subsets which may differentially respond to tumor-derived antigens. We have studied Treg cells in cancer in experimental mice that express naturally selected, polyclonal repertoire of CD4+ T cells and which preserve the heterogeneity of the Treg population. The majority of Treg cells present in healthy mice maintained a stable suppressor phenotype, expressed high level of Foxp3 and an exclusive set of TCRs not used by naive CD4+ T cells. A small Treg subset, utilized TCRs shared with effector T cells and expressed a lower level of Foxp3. We show that response to tumor-derived antigens induced efficient clonal recruitment and expansion of antigen-specific effector and Treg cells. However, the population of Treg cells in tumors was dominated by cells expressing TCRs shared with effector CD4+ T cells. In contrast, Treg cells expressing an exclusive set of TCRs, that dominate in healthy mice, accounted for only a small fraction of all Treg cells in tumor lesions. Our results suggest that the Treg repertoire in tumors is generated by conversion of effector CD4+ T cells or expansion of a minor subset of Treg cells. In conclusion, successful cancer immunotherapy may depend on the ability to block upregulation of Foxp3 in effector CD4+ T cells and/or selectively inhibiting the expansion of a minor Treg subset

Limit theorems for hybridization reactions on oligonucleotide microarrays

We derive herein the limiting laws for certain stationary distributions of birth-and-death processes related to the classical model of chemical adsorption-desorption reactions due to Langmuir. The model has been recently considered in the context of a hybridization reaction on an oligonucleotide DNA-microarray. Our results imply that the truncated-gamma- and beta-type distributions can be used as approximations to the observed distributions of the fluorescence readings of the oligo-probes on a microarray. These findings might be useful in developing new model-based, probe-specific methods of extracting target concentrations from array fluorescence readings.60F05 60G35 Birth-and-death process Density-dependent Markov process Oligonucleotide microarray Hybridization reaction Gamma distribution Langmuir adsorption-desorption model

Flow cytometry analysis of CD4⁺ T cells stimulated with dendritic cells presenting Ep63K peptide.

<p>Expression of Foxp3^GFP and CD25 is shown on gated CD4⁺ T cells. (A) Analysis of the CD4⁺ T cells from control (left) and draining (middle) lymph nodes of TCR^mini-Foxp3^GFP mice immunized with dendritic cells presenting Ep63K and CD4⁺ T cells isolated from TCR^mini-Foxp3^GFP mice and stimulated with dendritic cells <i>in vitro</i> (right). Dendritic cells (2×10⁵) were injected into footpads of TCR^mini-Foxp3^GFP mice for three days and mice were sacrificed after 5 days and popliteal lymph nodes were analyzed. (B) Analysis of gated CD4⁺ T cells isolated from tumors of tumor-bearing TCR^mini-Foxp3^GFP control mice (left) and mice immunized with dendritic cells (middle). TCR^mini-Foxp3^GFP mice were inoculated with B16 melanoma cells expressing NP-EP63K and at the same time dendritic cells were injected (5×10⁴). Injections of dendritic cells continued daily until mice were sacrificed. The abundance (%) of CD4⁺ T cells subsets CD25^-Foxp^GFP+, CD25⁺Foxp^GFP+ and CD25⁺Foxp^GFP- in the tumor tissue of TCR^mini-Foxp3^GFP control (dots) and immunized (stripes) mice. At least three mice were analyzed in each group.</p

Analysis of the TCR repertoires of naive, activated and T_reg cells in control and draining lymph nodes and tumor lesions of tumor-bearing TCR^mini-Foxp3^GFP mice.

<p>(A) Estimation of the TCR repertoire diversity using Chao mean. The data range spanned by vertical lines represent 95% confidence interval for Chao mean. The circles represent values of Chao estimator. (B) Estimation of the similarities of the TCR repertoires presented as a dendrogram based on the differences of the relative entropy against the pooled population for TCRs expressed by naive, activated (Activ.) and T_reg cells isolated from control (Ctrl. LN) and draining (Dr. LN) lymph nodes and tumor infiltrate (TILs). The dendrogram construction begins with each cell subset being a separate cluster. Then, the most similar cell populations (with the smallest difference in their relative entropies) are joined. We continue the process until we obtain a single cluster. The distance between two clusters is taken as the maximum difference in relative entropies of their members.</p

Flow cytometry, gene expression and functional analysis of effector and T_reg cells in TCR^mini-Foxp3^GFP mice bearing B16 melanoma tumors expressing NP-Ep63K.

<p>(A) Cells from control, draining lymph nodes and tumor infiltrates were isolated and single cell suspensions were stained with the relevant antibodies. Expression of Foxp3^GFP (left column) and CD25 (second column) on CD4⁺ T cells are shown. Expression of activation markers CD44 and CD62L is shown on gated effector CD4⁺ Foxp3⁻ cells (third column) and Foxp3 and CD25 expression are shown on gated CD4⁺ T cells (right column). Gates used to define Foxp3^GFP- cells (left column, continuous line) and subsets of naive CD44⁻CD62L⁺ (third column, continuous line) and activated CD44⁺CD62L⁻ (third column, dotted line) cells as well as Foxp3^GFPlo (left column, broken line) and Foxp3^GFPhi (left column, dotted line) T_reg cells for TCR repertoire studies are shown as rectangles. Naive cells were absent in tumors. Figure shows representative data of at least five mice analyzed. (B) Expression of GITR (left panels) and CTLA-4 (right panels) in CD4⁺Foxp3^GFP- (upper panels) and CD4⁺Foxp3^GFP+ (lower panels) cells isolated from tumor draining lymph nodes and tumors. Two individual mice were analyzed. (C) Analysis of Foxp3, IL-10 and TGF-β expression in Foxp3^GFP- and Foxp3^GFP+ CD4⁺ T cells isolated from tumors. Sorted cells were lysed directly and gene expression was detected by RT-PCR. Samples were normalized for β-actin expression. Two individual mice were analyzed. (D) Foxp3^GFP+CD4⁺ T cells isolated from the draining lymph nodes (DrLN) or tumors (TILs) suppress proliferation of effector CD4⁺ T cells. One of two experiments is shown.</p

Clonal abundance (%) of Ep63K-specific T cell clones in naive (sparse dots), activated (dense dots) and T_reg (stripes) subsets in the control and draining lymph nodes and tumors of tumor-bearing TCR^mini-Foxp3^GFP mice.

<p>The percentage of all Ep63K-specific clones in populations of naive, activated and T_reg cells is shown on each plot. Names of hybridomas expressing a particular TCRα chain are shown above upper panel. Asterisks indicate that no clones were found in the indicated cell subsets.</p