J Periodontal Implant Sci.  2017 Oct;47(5):292-311. 10.5051/jpis.2017.47.5.292.

Targeting the epitope spreader Pep19 by naïve human CD45RA⁺ regulatory T cells dictates a distinct suppressive T cell fate in a novel form of immunotherapy

• Kim HJ
• Cha GS
• Joo JY
• Lee J
• Kim SJ
• Lee J
• Park SY
• Choi J

• KMID: 2392717
• DOI: http://doi.org/10.5051/jpis.2017.47.5.292

Abstract

PURPOSE
Beyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance.
METHODS
Human polyclonal CD4⁺CD25⁺CD127(lo−) Tregs (127-Tregs) and naïve CD4⁺CD25⁺CD45RA⁺ Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an NOD/scid/IL-2Rγ(−/−) mouse model of collagen-induced arthritis.
RESULTS
Epitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of CD4⁺CD25⁺ Tregs at the articular joints in a mechanistic and orchestrated way.
CONCLUSIONS
We propose human naïve CD4⁺CD25⁺CD45RA⁺ Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis.

Keyword

Adoptive transfer; Autoimmune diseases; Heat-shock proteins; Immune tolerance; Regulatory T-lymphocytes; Rheumatoid arthritis

MeSH Terms

Adoptive Transfer
Animals
Apoptosis
Arthritis, Experimental
Arthritis, Rheumatoid
Autoimmune Diseases
Dendritic Cells
Epigenomics
Eragrostis
Heat-Shock Proteins
Humans*
Immune Tolerance
Immunotherapy*
In Vitro Techniques
Interleukin-2
Joints
Mice
Sensitivity and Specificity
Sirolimus
T-Lymphocytes
T-Lymphocytes, Regulatory*
Vaccination
Heat-Shock Proteins
Interleukin-2
Sirolimus

Figure

• 

  Figure 1 Fluorescence-activated cell sorting profiles of each representative subject demonstrating the expression of suppressive Treg markers — CD4, CD25, and Foxp3 — in ex vivo expanded 127-Tregs, 45RA-Tregs, Pep19-127-Tregs, and Pep19-45RA-Tregs. While a similar staining intensity was observed among 127-Tregs, 45RA-Tregs, and Pep19-127-Tregs, the highest intensity of these 3 phenotype markers was seen in Pep19-45RA-Tregs. Treg: regulatory T cell, Pep19: peptide 19, 127-Tregs: CD4+CD25+CD127lo− regulatory T cells, 45RA-Tregs: CD4+CD25+CD45RA+ regulatory T cells, Cy5: cyanine 5.

• 

  Figure 2 Fluorescence-activated cell sorting profiles of CD62L and CCR7 expression in the expanded Tregs of each representative subject. While 45RA-Tregs and Pep19-45RA-Tregs demonstrated a modest level of expression of CD62L and CCR7, 127-Tregs and Pep19-127-Tregs demonstrated a higher level of expression of these markers. Treg: regulatory T cell, 127-Tregs: CD4+CD25+CD127lo− regulatory T cells, Pep19: peptide 19, 45RA-Tregs: CD4+CD25+CD45RA+ regulatory T cells, Cy5: cyanine 5.

• 

  Figure 3 (A) Fluorescence-activated cell sorting profiles demonstrating the suppressive capability of expanded 127-Tregs, 45RA-Tregs, Pep19-127-Tregs, and Pep19-45RA-Tregs from each representative subject on the Teff proliferative response to Pep19 and the bystander self-antigens Hu19, Hu9, Hu14, and ox-LDL, as analyzed by the CFSE assay. (B) Bar graph demonstration of the mean±standard deviation of the fluorescence-activated cell sorting profiles of all subjects. Suppression of the Teff proliferative response to Pep19 and the various bystander self-antigens was most evident in Pep19-45RA-Tregs. 127-Tregs: CD4+CD25+CD127lo− regulatory T cells, 45RA-Tregs: CD4+CD25+CD45RA+ regulatory T cells, Pep19: peptide 19, CFSE: carboxyfluorescein diacetate succinimidyl ester, Hu: human, ox-LDL: oxidized low-density lipoprotein, Teff: effector T cell.

• 

  Figure 4 (A) Fluorescence-activated cell sorting profiles of expanded 127-Tregs, 45RA-Tregs, Pep19-127-Tregs, and Pep19-45RA-Tregs from each representative subject demonstrating a robust proliferative responses to Pep19 and the bystander self-antigens Hu19, Hu9, Hu14, and ox-LDL. Bar graph demonstration of the mean±standard deviation of the fluorescence-activated cell sorting profiles of all subjects. (B) The proliferative response of Pep19-45RA-Tregs was more pronounced than that of the 3 other kinds of Tregs. 127-Tregs: CD4+CD25+CD127lo− regulatory T cells, 45RA-Tregs: CD4+CD25+CD45RA+ regulatory T cells, Pep19: peptide 19, Treg: regulatory T cell, CFSE: carboxyfluorescein diacetate succinimidyl ester, Hu: human, ox-LDL: oxidized low-density lipoprotein.

• 

  Figure 5 Fluorescence-activated cell sorting profiles of CD95-mediated apoptosis in expanded Tregs of a representative subject after 24 hours of incubation. While 127-Tregs and Pep19-127-Tregs demonstrated a similar resistance to CD95L-mediated apoptosis, 45RA-Tregs and Pep19-45RA-Tregs exhibited slightly greater resistance. Treg: regulatory T cell, 127-Tregs: CD4+CD25+CD127lo− regulatory T cells, Pep19: peptide 19, 45RA-Tregs: CD4+CD25+CD45RA+ regulatory T cells, PI: propidium iodide, PE: phycoerythrin, FITC: fluorescein isothiocyanate.

• 

  Figure 6 Bar graph representation of the percentage of expanded Tregs in a representative subject expressing the Foxp3 protein and the percent of DNA demethylation in the Foxp3 TSDR. While a similar degree of DNA demethylation in the Foxp3 TSDR was among the Treg groups, the intensity of Foxp3 expression was most pronounced in Pep19-45RA-Tregs. Treg: regulatory T cell, TSDR: Treg-specific demethylation region, Pep19: peptide 19, 127-Tregs: CD4+CD25+CD127lo− regulatory T cells, 45RA-Tregs: CD4+CD25+CD45RA+ regulatory T cells. Statistically significant value, a)P<0.01; b)P<0.001.

• 

  Figure 7 (A) Bar graph representation of the severity of arthritis score of each mouse group on a scale of 0 to 3. (B) The lowest score was seen in the group III mice, which were infused with Pep19-45RA-Tregs. Representative micrographs of articular joints and cartilage stained with hematoxylin and eosin and safranin-O staining of articular cartilage in mice from groups I–III (bar=10 µm). (C) No evidence of proteoglycan loss from articular cartilage (a marker of cartilage destruction) was observed in the mice in groups I and III. The group II mice demonstrated a variable degree of proteoglycan loss from the articular cartilage. Representative micrographs of the immunohistochemical localization of CD4+CD25+ Tregs in articular joints in groups I–III. The homing pattern of CD4+CD25+ Tregs to the site of inflammation was most pronounced in the articular joints in group III (bar=50 µm). Pep19: peptide 19, 45RA-Tregs: CD4+CD25+CD45RA+ regulatory T cells, Treg: regulatory T cell.

• 

  Figure 8 (A) Photographic features demonstrating swollen footpads in a representative mouse from each group. (B) Line graphs depicting the changes in the mean thickness of the swollen footpad at each observation period. (C) Bar graph representing the mean paw thickness (±standard deviation) of each mouse group on day 38. Mice from both groups II and III showed minimal thickness, while the group I mice demonstrated greatest thickness. The value was significantly smaller in group III when compared with that of group I. (D) A diagrammatic plot of the mean survival rate of each mouse group during the observation period. The highest survival rate was seen in the group III mice. a)Statistically significant value (P<0.05).

• 

  Figure 9 (A) Expression of the Treg markers CD25 and Foxp3 by splenic CD4+ T cells isolated from a representative mouse from each group (I–III) in response to HSP60 from P. gingivalis, Pep19, Hu19, Hu9, Hu14, and ox-LDL as analyzed by fluorescence-activated cell sorting. (B) These Treg markers were variably expressed in response to the tested antigens in all 3 groups. Bar graph representation of the fluorescence-activated cell sorting profiles of CD25+Foxp3+ splenic Tregs in response to various bystander antigens. Statistically significant differences were noted at variable levels among the test groups except for the cognate epitope spreader antigen Pep19. Treg: regulatory T cell, Pep19: peptide 19, Hu19: peptide 19 of human HSP60, Hu9: peptide 9 of human HSP60, Hu14: peptide 14 of human HSP60, ox-LDL: oxidized low-density lipoprotein. Statistically significant value, a)P<0.05; b)P<0.01; c)P<0.001.

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