The pig (Sus scrofadomesticus) is an important experimental model for immunological studies as well as for study of porcine specific diseases¹. Phenotypic analysis of peripheral blood cells specifically T lymphocytes by surface marker staining in flow cytometry is widely used in present times for assessment of cell mediated immunity². Pig has a highly complex lymphocyte pool which includes cell subpopulations, such as peripheral CD4⁺CD8⁺ T cells, that are rare or absent in other species. Some of the peculiarities in pigs are the presence of double-positive T lymphocytes with the phenotype CD4⁺CD8low⁺ and CD4-CD8⁺(low)³. Thus, studies on dynamics of T-lymphocyte subpopulations are interesting. Importance of the pig immune system is increasingly felt in veterinary and human biomedical research because the pig has been recognized as a potential donor for xeno-transplantation. A lot of intrinsic and extrinsic factors like age, gut microflora, environmental factors, diseases etc. may influence lymphocyte sub-populations in blood⁴. The immune, neurological, and endocrine systems are other considerable regulatory factors, which may be involved in the lymphocyte subsets changes. Specific identification of the various subpopulations of leukocytes enables improved investigations of the immune response to various porcine pathogens such as Actinobacillus pleuropneumoniae, classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV) and Aujeszky disease virus⁵,⁶,⁷-⁸. Age related variations in lymphocyte subsets distribution in the peripheral blood have been reported to occur in pigs⁹. An understanding of dynamics of lymphocyte sub-populations in healthy pigs over a period of time is essential to ascertain and analyse the immune responses elicited by infectious agents and their vaccines.

Flow cytometry provides a rapid method to analyze normal and abnormal cell populations using a small sample volume and the results accurately reflect the in-vivo status of the populations¹⁰. Therefore, the aim of the present study was to analyse the changes in lymphocyte sub-populations in peripheral blood of healthy weaned piglets from 5 weeks to 36 weeks of age.

Variation in percentages of lymphocyte subpopulations in healthy weaned piglets from 5 to 36 weeks of age is shown in Fig. 2. The numerical data of percentage of various phenotypes of lymphocytes are presented in Table 1. The results of the study demonstrated that the overall relative percentage of CD3+ (total T lymphocytes %), CD3⁺CD4⁺CD8μ⁻ (T-helper cells %) and CD3⁺CD4⁻CD8^α+ (Cytotoxic T cells %), CD3⁺CD4⁺CD8^α+(low) (memory/activated T helper cells) increased with the age of the animal. Percentage of T lymphocytes (CD3+) sub population in total lymphocyte pool is increased from 43±2.5% to 61.9±3.9% between 5 weeks and 36 weeks of age, with a gradual rising pattern. The increase was statistically significant (P<0.05) from 14 weeks of age in comparison with 5 weeks of age. The increase in percentage of CD3⁺ cells was mostly due to CD3⁺CD4⁻CD8^α+ and CD3⁺CD4⁺CD8^α+.The percentage of CD3⁺CD4⁺CD8μ⁻ cells was relatively stable (ranging between 17.7±3.7% and 26.9±0.8) with mild and inconsistent increase from 18th week onwards. The percentage of cytotoxic T lymphocytes (CD3⁺CD4⁻CD8^α+) significantly (P<0.05) increased from 16.1±4.5% at 5 weeks to 42.0±1.6% at 36 weeks of age.

A gradual increase in double positive, memory/activated T helper cells (CD3⁺CD4⁺CD8a⁺(low)) was observed from 1.0±0.6% at 5 weeks to 7.2±0.3% at the end of the study. The ratio of T helper and cytotoxic T cells was around 1:1 at 5 weeks of age and gradually rose to 1:2 at about 36 weeks of age.

Inverse relationship with age was observed in relative NK cell population (CD3⁻CD4⁻CD8^α+), with a gradual decrease from 15^th week reaching up to 4.4±0.8% at 36 weeks of age. A decreasing trend in the probable B cell population (i.e. total lymphocytes minus (CD3+ cells plus NK cells) was observed between 14 and 32 weeks of age.

The present study documents the baseline data for percentage of the major lymphocyte sub-populations in PBL of healthy weaned piglets from 5 weeks to 36 weeks of age. T-lymphocyte subpopulations in swine are characterized primarily by cluster of differentiation (CD) marker phenotyping for CD3, CD4 and CD8. T-lymphocytes belong to the adaptive immune system and perform a wide array of functions in immune regulation, inflammation and protective immune responses¹². Porcine αβ T cells recognize antigens in a major histocompatibility complex (MHC)-restricted manner, whereas the αβ T-cells recognize antigens in a MHC non-restricted fashion. The CD4⁺CD8⁻ and CD4⁺CD8⁺low T-cell subsets of αβ T-cells recognize antigens presented in MHC class II molecules, while the CD4⁻CD8⁺ T-cell subset recognizes antigens presented in MHC class I molecules. Porcine CD8⁺αβT-cells are a prominent T-cell subset during antiviral responses, while porcine CD4⁺αβ T-cell responses predominantly occur in bacterial and parasitic infections. Porcine T-cell responses are also suppressed by some viruses and bacteria¹³. In the current study, the percentage of cytotoxic T lymphocytes (CD3⁺CD4⁻CD8^α+) increased about 2.5 times from 5 weeks to 36 weeks of age, while the percentage of CD3⁺CD4⁺CD8^μ-cells was relatively stable, with mild and inconsistent increase from 18 th week onwards. In a previous study, the absolute number of CD4⁺CD8⁻, CD4⁻CD8⁺ and CD4⁺CD8⁺ cells increased almost twice from birth till the 6th week of age. After this time, only CD4⁺CD8⁻ subset remained stable, while the number of CD4⁻CD8⁺ and CD4⁺CD8⁺ subsets gradually increased 1.5- and 2.5-fold from 6 weeks to 5 months of age for CD4⁻CD8⁺ and CD4⁺CD8⁺, respectively⁹. Hence, proper age matched controls is absolute necessityfor experiments to be conducted using pigs involving a longer duration of time. Pigs also have nearly twice as many CD8⁺ as CD4⁺T cells in the blood which is inverse compared to the ratio found in human and murine peripheral T cells¹⁴. Notably, swine have a significant population of mature (extrathymic) CD4/CD8 double-positive T cells that represent memory T cells³ and are thus speculated to play a role in memory immune responses. In this study, the percentage of double positive cells increased to about 7 times at 36 weeks of age as compared to 5 weeks of age. The results are in agreement with that obtained by Borghetti and others¹⁵, who investigated the age related changes in leukocyte counts of pigs till the 41^st day of life. The age-dependent increase in the amount of double positive lymphocytes seems to be the result of antigen-dependent maturation of naive CD4⁺ T helper lymphocytes to antigen-specific memory T helper cells. It might also be influenced by exposure to many external antigens as the age advances¹⁴.

Innate NK cells are the lymphocyte subpopulation known for their ability to provide the first line of defense against viral infections¹⁶. In pigs, a precise selection marker for phenotypic analysis of NK cells is not available; however, CD56 is used in some studies¹⁷,¹⁸. A comparable frequency of CD56⁺ and CD3⁻CD4⁻CD8^α+ cells in the pig PBMC was seen by Dwivedi and others¹³. There is paucity of information on age related changes in NK cell population in weaned piglets. Though not entirely comparable, a few studies in humans and rhesus monkeys indicated that the number of circulating CD56⁺CD16⁺ NK cells reduces with age¹⁹.

Proper phenotypic identification of porcine T lymphocytes is an important parameter in designing and evaluation of porcine immune responses. The results of the present study reiterates the need for use of age matched controls during experimental studies involving pigs, especially those requiring evaluation of lymphocytes and their responses to infection or vaccines.

The authors thank the Director, ICAR-NIHSAD, Bhopal for providing necessary facilities to carry out this work. Help extended by Dr. Richa Sood, Principal Scientist in animal sheds is duly acknowledged.

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